Inhibitors of nucleotidyl transferases and uses in herpes and hepatitis viral infections therefor

ABSTRACT

wherein the variables are as defined herein. Also provided are methods of treatment using agents so identified.

The present application is a divisional of U.S. application Ser. No.15/735,197, filed Dec. 10, 2017, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2016/036994, filed Jun. 10, 2016, which claims benefit of priorityto U.S. Provisional Application Ser. No. 62/309,303, filed Mar. 16,2016, and U.S. Provisional Application Ser. No. 62/174,385, filed Jun.11, 2015, the entire contents of each of which are hereby incorporatedby reference.

The invention was made with government support under Grant Nos. R01AI104494, U01 DK082871, and R03 AI109460 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND I. Field

The disclosure relates to the fields of pathology, virology, molecularbiology and pharmaceuticals. More specifically, the disclosure relatesto the identification of candidate inhibitors for the treatment andprevention of herpesvirus and hepatitis B diseases. Also provided arecompounds having such activity.

II. Related Art

Herpesviridae is a large family of DNA viruses that cause diseases invertebrates, including humans. These viruses are significant pathogensand, in addition to primary infections, cause latent, recurringinfections. At least six species of Herpesviridae—herpes simplex virus 1(HSV-1) and HSV-2 (both of which can cause orolabial herpes and genitalherpes), Varicella-zoster virus (which causes chickenpox and shingles),Epstein-Barr virus (which causes mononucleosis), Cytomegalovirus (whichcauses mental retardation and deafness in neonates), and Humanherpesvirus 6B (which causes roseola infantum and febrile seizures)—areextremely widespread among humans. More than 90% of adults have beeninfected with at least one of these, and a latent form of the virusremains in most people. Other viruses with human tropism include humanherpesvirus 6A, human herpesvirus 7 and Kaposi's sarcoma-associatedherpesvirus. There are more than 130 herpesviruses, including those thatinfect non-human mammals, birds, fish, reptiles, amphibians, andmollusks.

The drugs, acyclovir and ganciclovir, are considered the standardtreatments and prophylactic agents for infections caused by HSV, VZV andCMV. Until a decade ago, the impact of acyclovir on the control ofsevere and life-threatening herpesvirus infections was unprecedented.Recently, approval of new drugs (i.e., penciclovir and the oralprodrugs, valaciclovir, famciclovir, cidofovir, fomivirsen, andfoscarnet) has increased the number of therapeutic options for medicalpractitioners. Newer agents, such as brivudin and benzimidavir, are inongoing clinical development, while others have been suspended becauseof safety concerns. Regardless, new anti-herpes agents are needed toface clinical issues such as drug resistance, increased use ofanti-herpes prophylaxis, and safety concerns in small children orpregnant women.

Similarly, hepatitis B virus (HBV) is a hepatotropic DNA virus thatreplicates by reverse transcription (Hostomsky et al., 1993). Itchronically infects >350 million people world-wide and kills up to 1.2million patients annually by inducing liver failure and liver cancer(Steitz, 1995; Katayanagi et al., 1990; Yang et al., 1990; Lai et al.,2000). Reverse transcription is catalyzed by a virally-encodedpolymerase that has two enzymatic activities: a DNA polymerase thatsynthesizes new DNA and a ribonuclease H (RNAseH) that destroys theviral RNA after it has been copied into DNA (Hostomsky et al., 1993;Rice et al., 2001; Hickman et al., 1994; Ariyoshi et al., 1994). Bothactivities are essential for viral replication.

HBV infections are treated with interferon α or one of fivenucleos(t)ide analogs (Parker et al., 2004; Song et al., 2004; Lima etal., 2001). Interferon α leads to sustained clinical improvement in20-30% of patients, but the infection is very rarely cleared (Hostomskyet al., 1993; Katayanagi et al., 1990; Braunshofer-Reiter et al., 1998).The nucleos(t)ide analogs are used more frequently than interferon. Theyinhibit DNA synthesis and suppress viral replication by 4-5 log₁₀ in upto 70-90% patients, often to below the standard clinical detection limitof 300-400 copies/ml (Braunshofer-Reiter et al., 1998; Nowotny et al.,2005; Klumpp et al., 2003. However, treatment eradicates the infectionas measured by loss of the viral surface antigen (HBsAg) from the serumin only 3-6% of patients even after years of therapy (Braunshofer-Reiteret al., 1998; Nowotny et al., 2005; Klumpp et al., 2003; Nowotny et al.,2006). Antiviral resistance was a major problem with the earliernucleos(t)ide analogs, but resistance to the newer drugs entecavir andtenofovir is very low (Parker et al., 2004; Keck et al., 1998; Goedkenet al., 2001; Li et al., 1995). This has converted HBV from a steadilyworsening disease into a controllable condition for most individuals(McClure, 1993). The cost of this control is indefinite administrationof the drugs (probably life-long; (Song et al., 2004), with ongoingexpenses of $400-600/month (Poch et al., 1989; Hu et al. 1996; Hu etal., 1997) and unpredictable adverse effects associated withdecades-long exposure to the drugs.

As such, there remains a need to develop new therapeutic options forthese diseases.

SUMMARY

Thus, in accordance with the present disclosure, there is provided amethod of inhibiting a cellular or herpesvirus nucleic acid metabolismenzyme comprising contacting said enzyme with a compound having theformula:

or a compound of the formula:

wherein:

-   -   R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₅ and R₈ are each independently hydrogen, alkyl_((C≤8)), or        substituted alkyl_((C≤8));    -   R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substituted        alkyl_((C≤8)); and    -   R₇ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), or a        substituted version of any of these groups; or a compound of the        formula:

wherein:

-   -   R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));        and    -   R₁₁ is hydrogen or Y₁—O—X₁—OR₁₂; wherein:        -   Y₁ is alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8));        -   X₁ is arenediyl_((C≤12)), heteroarenediyl_((C≤12)), or a            substituted version of either of these groups;        -   R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)),            or a substituted version of any of these groups;            or a pharmaceutically acceptable salt or tautomer thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₅ and R₈ are each independently hydrogen, alkyl_((C≤8)), or        substituted alkyl_((C≤8));    -   R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substituted        alkyl_((C≤8)); and    -   R₇ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), or a        substituted version of any of these groups; or        or a pharmaceutically acceptable salt or tautomer thereof. In        other embodiments, the compound is further defined as:

wherein:

-   -   R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));        and    -   R₁₁ is hydrogen or Y₁—O—X₁—OR₁₂; wherein:        -   Y₁ is alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8));        -   X₁ is arenediyl_((C≤12)), heteroarenediyl_((C≤12)), or a            substituted version of either of these groups;        -   R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)),            or a substituted version of any of these groups;            or a pharmaceutically acceptable salt or tautomer thereof.

In other embodiments, the compound is further defined as:

or a pharmaceutically acceptable salt or tautomer thereof. In otherembodiments, the compound is further defined as:

or a pharmaceutically acceptable salt or tautomer thereof.

The salt maybe an ethanolamine salt. The method may further comprisecontacting said enzyme with a second inhibitor of said enzyme, orfurther comprise contacting said enzyme with said compound a secondtime. The enzyme may be located in a cell, which cell may be located invitro or located in a living subject. The subject may be a vertebrateinfected with a herpesvirus. The compound may be administeredintravenously, intra-arterially, ocularly, orally, buccally, nasally,rectally, vaginally, topically, intramuscularly, intradermally,cutaneously or subcutaneously. The subject may be further administered asecond anti-herpesvirus therapy distinct from the compound. The secondanti-herpesvirus therapy may be foscarnet or a nucleoside analog, suchas acyclovir, famciclovir, valaciclovir, penciclovir, or ganciclovir.The second anti-herpesvirus therapy may be administered to the subjectbefore or after said compound. The second anti-herpesvirus therapy maybe administered to said subject at the same time as said compound.

The subject may have previously received a first-line anti-herpesvirustherapy, and further may have developed resistance to said first-lineanti-herpesvirus therapy. The herpevirus may be selected from a humanalpha herpesvirus, a human beta herpesvirus or a human gammaherpesvirus. The human alpha herpesvirus may be selected from herpessimplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), andVaricella-Zoster virus (VZV). The human beta herpesvirus may be selectedfrom human cytomegalovirus (HCMV), human herpesvirus 6A, (HHV-6A), humanherpesvirus 6B (HHV-6B), and human herpesvirus 7 (HHV-7). The humangamma herpesvirus may be selected from Epstein-Barr virus (EBV) andKaposi's sarcoma herpesvirus (KSHV). The herpesvirus may be a non-humanherpesvirus, such as Marek's disease virus, equine herpesviruses, Bovineherpeviruses, or pseudorabies virus.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the formula:

or a pharmaceutically acceptable salt or tautomer thereof. The salt maybe an ethanolamine salt. In some embodiments, the compound is dispersedin a pharmaceutically acceptable buffer, diluent, excipient or carrier.In some embodiments, the pharmaceutical composition is formulated foradministration: orally, nasally, ocularly, buccally, corneally,rectally, vaginally, or topically. In other embodiments, thepharmaceutical composition formulated for administration via injection.In some embodiments, the injection is formulated for administration:intradermally, cutaneously, subcutaneously, intramuscularly,intraperitoneally, intraarterially, or intravenously.

In still another aspect, the present disclosure provides compounds ofthe formula:

wherein:

-   -   R₁ is aryl_((C≤12)), aralkyl_((C≤18)), heteroaryl_((C≤12)),        alkylamino_((C≤12)), dialkylamino_((C≤12)), arylamino_((C≤12)),        diarylamino_((C≤12)), aralkylamino_((C≤18)),        diaralkylamino_((C≤18)), or a substituted version of any of        these groups;    -   R₂ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and    -   R₃ is hydrogen, amino, carboxyl, cyano, halo, hydroxy, nitro,        hydroxysulfonyl, or sulfonylamine; or        -   alkyl_((C≤8)), aryl_((C≤8)), acyl_((C≤8)), alkoxy_((C≤8)),            acyloxy_((C≤8)), amido_((C≤8)), or substituted version of            any of these groups;    -   X₂ is hydrogen or —C(O)R_(a), wherein: R_(a) is hydroxy,        alkoxy_((C≤8)), or substituted alkoxy_((C≤8)); or compounds of        the formula:

wherein:

-   -   R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₅ and R₈ are each independently hydrogen, alkyl_((C≤8)), or        substituted alkyl_((C≤8));    -   R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substituted        alkyl_((C≤8)); and    -   R₇ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), or a        substituted version of any of these groups; or        compounds of the formula:

wherein:

-   -   R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));        and    -   R₁₁ is hydrogen or Y₁—O—X₁—OR₁₂; wherein:        -   Y₁ is alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8));        -   X₁ is arenediyl_((C≤12)), heteroarenediyl_((C≤12)), or a            substituted version of either of these groups;        -   R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)),            or a substituted version of any of these groups;            or a pharmaceutically acceptable salt thereof. In some            embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is aryl_((C≤12)), aralkyl_((C≤18)), heteroaryl_((C≤12)),        alkylamino_((C≤12)), dialkylamino_((C≤12)), arylamino_((C≤12)),        diarylamino_((C≤12)), aralkylamino_((C≤18)),        diaralkylamino_((C≤18)), or a substituted version of any of        these groups; and    -   R₂ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));    -   X₂ is hydrogen or —C(O)R_(a), wherein: R_(a) is hydroxy,        alkoxy_((C≤8)), or substituted alkoxy_((C≤8));        or a pharmaceutically acceptable salt thereof. In other        embodiments, the compounds are further defined as:

wherein:

-   -   R₅ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));    -   R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substituted        alkyl_((C≤8)); and    -   R₇ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), or a        substituted version of any of these groups;        or a pharmaceutically acceptable salt thereof. In other        embodiments, the compounds are further defined as:

wherein:

-   -   R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));        and    -   R₁₁ is hydrogen or Y₁—O—X₁—OR₁₂; wherein:        -   Y₁ is alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8));        -   X₁ is arenediyl_((C≤12)), heteroarenediyl_((C≤12)), or a            substituted version of either of these groups;        -   R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)),            or a substituted version of any of these groups;            or a pharmaceutically acceptable salt thereof.

In some embodiments, R₂, R₅, or R₁₀ is hydrogen. In some embodiments,R₂, R₅, and R₁₀ are hydrogen. In some embodiments, R₁ isaralky_((C≤18)), aralkylamino_((C≤18)), or a substituted version ofeither group. In some embodiments, R₆ is hydroxy. In some embodiments,R₇ is aryl_((C≤12)). In some embodiments, R₁₁ is Y₁—O—X₁—OR₁₂; wherein:

Y₁ is alkanediyl_((C≤8));

X₁ is arenediyl_((C≤12)), or a substituted version of either of thesegroups;

R₁₂ is aryl_((C≤12)) or substituted aryl_((C≤12)).

In some embodiments, the compounds are further defined as:

or a pharmaceutically acceptable salt thereof.

In still yet another aspect, the present disclosure provides a compoundof the formula:

or a pharmaceutically acceptable salt thereof.

In still yet another aspect, the present disclosure providespharmaceutical compositions comprising:

(A) a compound described herein; and

(B) an excipient.

In some embodiments, the pharmaceutical composition is formulated foradministration: orally, nasally, buccally, corneally, rectally,vaginally, topically, intradermally, cutaneously, ocularly,subcutaneously, intramuscularly, intraperitoneally, intraarterially, orintravenously. In some embodiments, the pharmaceutical composition isformulated as a unit dose.

In yet another aspect, the present disclosure provides methods ofinhibiting a hepatitis B virus RNaseH comprising administering aneffective amount of a compound or composition described herein. In someembodiments, the method is performed in vitro. In other embodiments, themethod is performed in vivo. In other embodiments, the method isperformed ex vivo. In some embodiments, the methods are sufficient toinhibit viral replication.

In still yet another aspect, the present disclosure provides methods ofinhibiting replication of a hepatitis B virus comprising contacting thevirus with an effective amount of a compound or composition describedherein. In some embodiments, the method is performed in vitro. In otherembodiments, the method is performed in vivo. In other embodiments, themethod is performed ex vivo. In some embodiments, the methods aresufficient to treat an infection of a hepatitis B virus.

In another aspect, the present disclosure provides methods of treatingan infection of a hepatitis B virus in a patient comprisingadministering a therapeutically effective amount of a compound orcomposition described herein. In some embodiments, the method furthercomprises a second antiviral treatment. In some embodiments, the secondantiviral therapy is interferon alfa-2b, lamivudine, adefovir,telbivudine, entercavir, or tenofovir. In some embodiments, the patientis a mammal such as a human.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects, or +/−5% of the stated value.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1. Structure of Piroctone Olamine.

FIG. 2. Compound #191 inhibits replication of HSV-2 primary clinicalisolates. Vero cell monolayers were infected with the indicated patientisolates at moi of 0.1 in the presence of piroctone olamine (#191) orDMSO control at 5 μM. Cultures were collected 24 hours post-infectionand infectious virus titers were determined by plaque assay. Titers in#191-treated samples were subtracted from DMSO-treated samples. Valuesare the averages of duplicate cultures.

FIG. 3. Sensitivity of thymidine kinase-deficient HSV-2 to piroctoneolamine. Vero cell monolayers were infected with the indicated wild-typeor mutant HSV-1 or HSV-2 strains at moi of 0.1 in the presence of DMSOcontrol or piroctone olamine at 5 μM. Duplicate cultures were collected24 hours post-infection and infectious virus titers were determined byplaque assay. Titers in compound-treated samples were subtracted fromDMSO-treated samples. Values are the averages±one standard error of themean.

FIGS. 4A & 4B. RNAseH inhibitors work synergistically with Lamivudineagainst HBV replication. Chou-Talaly combination indexes for compounds#1 (FIG. 4A) and #46 (FIG. 4B) together with Lamivudine. Additiveinteractions are shown with the red line, synergistic interactions belowthe line, and antagonistic interactions are above the line.

FIGS. 5A & 5B. HBV's genetic variation is unlikely to present a barrierto RNAseH drug development. Four variant patient-derived RNAseH enzymeswere expressed as recombinant enzymes, purified, and tested in an RNAseHassay with compounds #1 (FIG. 5A) and #46 (FIG. 5B) at their respectiveIC₅₀s.

DETAILED DESCRIPTION

The inventors have previously demonstrated that inhibitors ofnucleotidyl-transferase superfamily (NTS) enzymes can come from multipledifferent chemical classes. The compounds described herein may be usedfrom a variety of different viral infections including herpesvirus andhepatitis B virus. Inhibitors of NTS enzymes like this compound may wellhave a high barrier to development of antiviral resistance, and itsunique mode of action suggests that it should be a good candidate forcombination therapy with the existing antiviral drugs to improve overallefficacy of antiviral therapy. These and other aspects of the disclosureare discussed in detail below.

A. HERPESVIRUS

Herpesviruses are a diverse group of enveloped viruses having a large,double-stranded DNA genome enclosed in an icosahedral capsid (Pellet &Roizman 2013). The herpesviruses rely on the host cell RNA polymerase IIfor transcription, but encode all of the enzymes needed for replicationof their genomes, including DNA polymerase, helicase, primase,terminase, ribonucleotide reductase, and thymidine kinase. Allherpesviruses share the capacity to establish latency in host cells,allowing them to maintain the infection for the life of the host.Periodic reactivation from latency in response to cues in the cellularenvironment leads to lytic replication at mucosal surfaces, causingrecurrent disease and providing the opportunity for transmission touninfected individuals.

The herpesviruses are divided into three subclasses based primarily ontheir cellular tropism and characteristics of the latent infection. Thehuman alpha herpesviruses herpes simplex virus 1 (HSV-1) (Roizman etal., 2013), herpes simplex virus 2 (HSV-2) (Roizman et al., 2013) andVaricella-Zoster virus (VZV) (Arvin & Gilden 2013) establish latency insensory neurons where they may remain quiescent for long periods oftime. HSV-1 and HSV-2 are similar viruses with colinear genomes and 83%nucleotide sequence identity in protein coding regions (Dolan et al.,1998); VZV contains a smaller, less homologous genome. The human betaherpesviruses human cytomegalovirus (HCMV), human herpesvirus 6A(HHV-6A), human herpesvirus 6B (HHV-6B) and human herpesvirus 7 (HHV-7)(Yamanishi et al., 2013) establish latency predominantly in mononuclearcells. The human gamma herpesviruses Epstein-Barr virus (EBV)(Longnecker et al., 2013) and Kaposi's sarcoma herpesvirus (KSHV)(Damania & Cesarman 2013) stimulate cellular proliferation uponinfection. EBV infects B lymphocytes, where it establishes latency, andalso epithelial cells. By contrast, endothelial cells harbor the latentreservoir of KSHV, although the virus infects numerous other cell typesas well. The genomes of latent beta and gamma herpesviruses arereplicated as the host cell divides in order to maintain latentinfection.

Herpesviruses related the human alpha, beta and gamma herpesvirusesinfect numerous animal species, including several of significanteconomic importance. Key among these are pseudorabies virus whichinfects pigs, Marek's disease virus which infects chickens, bovineherpesvirus, equine herpesvirus, and salmonid and related herpesvirusesthat infect game fish.

1. Pathology

Primary infections with herpesviruses produce a broad spectrum ofdisease. HSV-1 causes numerous maladies (Roizman et al., 2013):gingivostomatitis; eczema herpeticum; herpes gladiatorum; less commonbut frequently fatal encephalitis; and an increasing proportion ofulcerative anogential lesions (Gilbert et al., 2011; Horowitz et al.,2011; Pena et al., 2010; Smith & Roberts 2009). Nearly two-thirds of theU.S. population has been exposed to HSV-1 (Xu et al., 2006). HSV-2infects approximately 17% of Americans (Xu et al., 2006) and up to 75%of some demographics world-wide (Obasi et al., 1999 and Kamali et al.,1999), with an estimated global disease burden of more than half abillion people (Looker, et al., 2008). HSV-2 is the primary cause ofulcerative anogenital lesions. In addition, HSV-1 and HSV-2 may betransmitted from a pregnant woman to her child during birth, oftencausing potentially fatal disseminated disease in the newborn (Kimberlin2007). HCMV is the most common in utero virus infection (Manicklal etal., 2013), and approximately 8,000 HCMV-infected infants born each yearin the U.S. suffer sensorineural deafness, chorioretinitis, and/ormental retardation (James et al., 2009). In immunocompromisedindividuals, HCMV can cause mononucleosis, retinitis, colitis,pneumonitis, and esophagitis. These serious HCMV infections areassociated with increased morbidity and mortality (Komatsu et al.,2014). EBV causes the vast majority of infectious mononucleosis, whichstrikes nearly half of young adults (Luzuriaga & Sullivan 2010).Notably, of the eight human herpesviruses, a vaccine is available onlyfor VZV.

The novel capacity of herpesviruses to establish and reactivate fromlatency is also associated with numerous pathologies. HSV-1 causesrecurrent cold sores; a significant proportion of devastating viralencephalitis; and corneal scarring known as herpetic stromal keratitiswhich is the most frequent infectious cause of blindness, afflictingnearly 400,000 persons annually in the U.S. (Roizman et al., 2013).HSV-2 frequently reactivates to cause genital ulcers and prior HSV-2infection is associated with an increased risk of human immunodeficiencyvirus (HIV) acquisition (Roizman et al., 2013). Infants who surviveHSV-1 or HSV-2 infections often experience life-long sequellae andperiodic recurrent lesions (Kimberlin 2007 and James et al., 2009). VZVreactivates in up to half of older adults (Cohen 2013), and painassociated with the classic Zoster (shingles) rash and post-rashneuralgia can be excruciating. HCMV reactivation is associated withincreased incidence of restenosis after angioplasty (Popovic et al.,2012), and also causes significant morbidity and mortality in recipientsof bone marrow and solid organ transplants (Snydman 2008). Latent EBVinfection is associated with a variety of cancers including Burkitt'slymphoma, two types of Hodgkin's lymphoma, non-Hodgkin's lymphoma,nasopharyngeal carcinoma, and post-transplant lymphoproliferativedisease. Latent KSHV infection can lead to three types of cancer:Kaposi's sarcoma, pleural effusion lymphoma, and Castleman's disease(Damania & Cesarman 2013).

Veterinary herpesviruses also take a significant toll on livestock.Marek's disease is highly contagious, spreading rapidly through flocksof chickens that have not been vaccinated. It causes T cell lymphomawith infiltration of nerves and somatic organs, leading to paralysis anddeath in up to 80% of infected birds (Hirari, 2001). In addition,vaccine efficacy has declined with a concomitant increase in Marek'svirus virulence (Gimeno, 2008). Pseudorabies (PRV) is the second mosteconomically important viral disease of swine. Although PRV does notcause illness in adult swine, infection of pregnant sows results in ahigh incidence of abortion or resorption (Smith, 1997). Piglets infectedwith PRV suffer coughing, sneezing, fever, constipation, and a varietyof neurologic symptoms. Mortality in piglets less than one month of ageis close to 100%, but declines rapidly with age (Nauwynck et al., 2007).Ruminants and dogs and cats are also susceptible to lethal PRV infection(Fenner et al., 1993). In cattle, symptoms include intense itchingfollowed by neurological signs and death. In dogs, intense itching isaccompanied by jaw and pharyngeal paralysis and subsequent death (Decaroet al., 2008). In cats, usually no symptoms are observed because thedisease is so rapidly fatal (Gaskell et al., 2007). Bovine herpesviruses(BHV) cause a variety of illnesses in young cattle, and can also causeabortion. Although the illnesses caused by BHV's are mostly notlife-threatening, they cause important diseases because infection maytrigger a decline in meat and milk production and affect traderestrictions (Nandi et al., 2009). Equine herpesviruses typically causerespiratory disease, but certain species also cause myeloencephalopathyin horses, abortion and occasionally neonatal mortality due to pneumonia(Fortier et al., 2010). The herpesviruses of various fish species cancause significant mortality in aquaculture settings, particularly at thefingerling stage (Hanson et al., 2011). Importantly, all of theseviruses share the same basic genomic replication mechanisms, so if thepresumed mechanism by which the NTS enzymes inhibit HSV-1 and HSV-2 iscorrect, most of the other herpesvirus pathogens should also be highlysensitive to NTS inhibitors. Development of NTS inhibitors intoanti-herpesvirus drugs would be particularly valuable in cases likeHCMV, where current antiviral therapies frequently drive resistance andare plagued by toxicity issues (Weller and Kuchta, 2013). Finally, NTSinhibitors may be promising candidates for pan anti-herpesvirus drugdevelopment due to similarities in replication mechanisms of all theherpesviruses.

2. Infection and Latency

Enveloped herpesvirus particles fuse with the plasma membrane of a cell,releasing viral regulatory proteins and the viral capsid containing thelinear double-stranded DNA genome into the cytoplasm. The capsidsdeliver the viral genome to the nucleus via release through nuclearpores, whereupon the genome circularizes and becomes transcriptionallyactive. Viral infection at this point can proceed by two patterns, lyticor latent. In the lytic cycle, coordinated phases of viral transcriptionlead to expression of the viral regulatory proteins, viral enzymes, andconcurrently with the onset of DNA replication, the viral structuralproteins. Nascent viral capsids assemble in the nucleus and then budthrough the nuclear membranes to acquire their envelope (Mettenleiter etal., 2009). Release from the cells is primarily lytic, resulting in thedeath of the cell. Alternatively, the virus may enter a latent state,where transcription is limited to a few viral regulatory loci and viralDNA replication is strictly limited. Upon recognition of appropriatecellular stimuli, viral transcription reverts to the lytic pattern andproductive viral replication occurs.

Initial infections with alpha herpesviruses are lytic, resulting indispersion of the virus to other cells and organs. These virusesestablish latency in the unique environment of the neuron, and also insatellite cells in the case of VZV. During latency, replication of alphaherpesvirus DNA may occur at a low level because latently infectedneurons contain multiple copies of the genome (Chen et al., 2002; Wanget al., 2005). Once latency is established, DNA replication increasesmarkedly only during a reactivation event. Initial infections with betaherpesviruses are typically non-lytic but may cause cell-cell fusion.The gamma herpesviruses stimulate proliferation of infected cells,replicating their DNA along with cellular DNA replication to transmitcopies of the viral genome to daughter cells (Longnecker et al., 2013).All the herpesviruses cause episodic lytic infection of at least somecell types, allowing them to be shed from mucosal surfaces to facilitatetransmission to uninfected individuals.

3. Genomic Replication

Circularization of the linear double-stranded herpesvirus DNA occurs inthe nucleus shortly after viral uncoating, presumably through arecombination-mediated event. Replication of the viral DNA occurs in thenucleus within three-dimensional domains termed replication compartments(Quinlan et al., 1984). DNA replication is thought to employ adouble-stranded rolling circle mechanism [reviewed in (Weller & Coen2012; Lehman & Boehmer 1999)]. In preparation for viral DNA replication,virus-encoded transcriptional activators upregulate expression ofproteins involved in nucleic acid metabolism. DNA replication theninitiates at one of three viral origins of DNA replication and ismediated through action of the viral ICP6 origin binding protein. (Allviral gene names in this section are for HSV-1). DNA synthesis is primedby the viral helicase/primase complex (pUL5, pUL8, and pUL52). DNAelongation occurs by coupled leading- and lagging-strand DNA synthesisthrough formation of a replication fork that is grossly similar to theforks that replicate cellular DNA. DNA synthesis is catalyzed by thepUL30 DNA polymerase/UL42 processivity protein complex that alsopossesses 5′-3′ exonuclease, 3′-5′ exonuclease, and RNase H activities.Helical torsion is relieved by the viral helicase/primase complex, andproper replication fork initiation, architecture and dynamics arepromoted by the ICP8 single-stranded DNA binding protein. The initialproduct of DNA replication is a head-to-tail concatamer, but later inthe replication cycle complex branched concatamers accumulate throughrecombination and/or re-initiation mechanisms. The concatamer is cleavedto unit length by the terminase complex (pUL15, pUL28, and pUL33)(Selvarajan et al., 2013) during encapsidation of the viral genome intopre-formed viral capsids. Without wishing to be bound by any theory, itis believed that some of the compounds described herein inhibit theactivity of pUL15 terminase.

4. Therapeutic Targets in the Herpesvirus Genome

Possible Targets for Nucleotidyl Transferase Superfamily Inhibitors inthe Herpesvirus Genomes.

The inhibitors screened here function against HIV by binding to theviral RNase H or integrase active sites and chelating the essentialdivalent cations within the active site (Fuji et al., 2009; Su et al.,2010; Chung et al., 2011; Billamboz et al., 2011; Himmel et al., 2009;Kirschberg et al., 2009). Other compounds screened here are chemicallyrelated to inhibitors of the HIV RNase H and integrase. Therefore, theirpresumed mechanism of action is to inhibit one or more of the viraland/or cellular NTS enzymes essential for herpesviral genomicreplication. This mechanism has not yet been tested.

For the herpes simplex viruses, candidate genes include the RNase Hactivity of the pUL30 DNA polymerase (Liu et al., 2006), the 3′-5′exonuclease activity of pUL30 (Coen 1996), the strand transfer activityof ICP8 (Bortner et al., 1993; Nimonkar & Boehmer, 2003), or the 5′-3′exonuclease activity of the pUL12 polymerase accessory protein(Schumacher et al., 2012) that are directly involved in virusreplication (Weller & Coen 2012). The pUL15 terminase protein thatcleaves the concatameric viral DNA produced by DNA replication into themature linear monomers is also a prime candidate (Selvarajan et al.,2013).

Other herpesviruses encode proteins with functions consistent with NTSenzymes that could be plausible targets. For example, pUL98 is the HCMVortholog of HSV pUL12 and is functionally conserved, as demonstrated bytrans-complementation experiments (Gao et al., 1998). At least two ofthe seven HCMV proteins involved in encapsidation form an essentialterminase complex which likely functions as both an endonuclease and aDNA translocase during DNA cleavage and packaging (Bogner, 2002; Hwang &Bogner, 2002; Scheffczik et al., 2002; Scholz et al., 2003). These genesare conserved throughout the herpesvirus family (Alba et al., 2001) anddeletion of any of the seven results in accumulation of empty capsids inthe nucleus. The human cytomegalovirus (HCMV) terminase subunits pUL56and pUL89, encoded by the UL56 and UL89 genes, have been extensivelystudied. Both gene products form toroidal structures, bind DNA, and havenuclease activity (Bogner et al., 1998; Scheffczik et al., 2002). WhilepUL56 mediates the specific binding to pac sequences on DNA concatamersand provides energy and structural assistance for DNA translocation intothe procapsids, pUL89 cleaves the DNA concatomers (Bogner, 2002). Theseare the orthologs of HSV terminase subunits pUL15 and pUL28.

Cellular proteins are also plausible targets for the action of the NTSinhibitors, especially because DNA recombination events appear to beimportant during productive replication (Weller & Coen 2012). Theseproteins include the human RNase H1 that could assist in removal of RNAprimers for DNA synthesis. Other candidates include the Fen1endonuclease that may assist in removal of primers (Zhu et al., 2010),and the double-stranded break repair enzymes Mre11, Rad50, NBS1, Rad51(Weizman & Weller 2011), and Rad52 (Schumacher et al., 2012). Thebase-excision repair enzymes SSH2 and MLH1 which form complexes that arerecruited to viral replication sites and contribute to HSV genomicreplication (Mohni et al., 2011) are also plausible targets.

5. Treatments

Herpesvirus DNA polymerase inhibitors (nucleoside analogs), includingacyclovir, famciclovir, valaciclovir, penciclovir, and ganciclovir arethe most common forms of treatment. A pyrophosphate analog, foscarnet,also inhibits the herpesvirus DNA polymerases. A DNA helicase-primaseinhibitor, AIC316 (pritelivir), was shown to reduce HSV-2 shedding andnumber of days without lesions in a phase 2 clinical trial (Wald et al.,2014). However, a subsequent double-blind trial by the same group wasterminated by the sponsor because of concurrent findings of toxicity inmonkeys (De et al., 2015). Similarly, the helicase-primase inhibitorAmenamevir (ASP2151) is active against HSV-1 and HSV-2 in culture (Chonoet al., 2010) and significantly reduced the median time to lesionhealing in a phase II clinical trial (Tyring et al., 2012), but asubsequent trial was terminated due to adverse effects (De et al.,2015). CMX001 (brincidofovir), an orally bioavailable lipid conjugate ofcidofovir, potentiates the antiviral effect of acyclovir in miceinoculated intranasally with HSV-1 or HSV-2 (Prichard et al., 2011).N-Methanocarbathymidine (N-MCT) reduces lethality in a mouse model ofHSV-2 infection (Quenelle et al., 2011) and a guinea pig model ofneonatal herpes (Bernstein et al., 2011). N-MCT also reduces acute andrecurrent disease caused by HSV-2 in an adult guinea pig model. Themonoamine oxidase inhibitor tranylcypromine (TCP), which also blocks theactivity of histone demethylase LSD1, reduces HSV-1 infection of thecornea, trigeminal ganglia and brain of mice, corneal disease, andpercentage of mice shedding virus upon induced reactivation (Yao et al.,2014). TCP has also been tested in a rabbit eye model of recurrentinfection with HSV-1 and the mouse and guinea pig models of HSV-2genital infection. An acyclic nucleoside phosphonate, PMEO-DAPym,inhibits HSV replication in a variety of cultured cell types bytargeting the viral DNA polymerase (Balzarini et al., 2013). The HIVintegrase inhibitor, Raltegravir, has a small amount of inhibitoryactivity against replication of several herpesviruses in cultured cells(Zhou et al., 2014; Yan et al., 2014) and appears to target thepolymerase processivity factor UL42 (Zhou et al., 2014). Two otherintegrase inhibitors, XZ15 and XZ45, reduce replication of HSV-1 in cellculture by approximately 800- to 8000-fold, respectively (Yan et al.,2014). XZ45 also inhibits HCMV replication and KSHV gene expression (Yanet al., 2014).

Therapy based on existing drugs such as acyclovir is incompletelyeffective (Johnston et al., 2012), and viral resistance to currentnucleos(t)ide analog therapies is relatively common. Acyclovir resistantvariants are particularly prevalent among children, theimmunocompromised, and patients with herpetic stromal keratitis (Duan etal., 2008; Wang et al., 2011; Field & Vere Hodge, 2013; Morfin &Thouvenot, 2003; Andrei & Snoeck, 2013). Ganciclovir-resistant variantsoccur in the naturally circulating viral population (Drew et al., 1993)and can be selected in patients over time (Marfori et al., 2007; Imai etal., 2004; Drew et al., 2001; Drew et al., 1999).

B. HEPATITIS B VIRUS

1. Biology

Hepatitis B virus, abbreviated HBV, is a species of the genusOrthohepadnavirus, which is likewise a part of the Hepadnaviridae familyof viruses. This virus causes the disease hepatitis B. In addition tocausing hepatitis B, infection with HBV can lead to hepatic fibrosis,cirrhosis and hepatocellular carcinoma. It has also been suggested thatit may increase the risk of pancreatic cancer.

The hepatitis B virus is classified as the type species of theOrthohepadnavirus, which contains at least five other species: thepomona roundleaf bat hepatitis virus, long-fingered bat hepatitis virus,the Ground squirrel hepatitis virus, Woodchuck hepatitis virus, and theWoolly monkey hepatitis B virus. The genus is classified as part of theHepadnaviridae family along with Avihepadnavirus. This family of viruseshave not been assigned to a viral order. Viruses similar to hepatitis Bhave been found in all the Old World apes (orangutan, gibbons, gorillasand chimpanzees) and from a New World woolly monkey suggesting anancient origin for this virus in primates.

The virus is divided into four major serotypes (adr, adw, ayr, ayw)based on antigenic epitopes present on its envelope proteins, and intoeight genotypes (A-H) according to overall nucleotide sequence variationof the genome. The genotypes have a distinct geographical distributionand are used in tracing the evolution and transmission of the virus.Differences between genotypes affect the disease severity, course andlikelihood of complications, and response to treatment and possiblyvaccination.

The virus particle (virion) consists of an outer lipid envelope and anicosahedral nucleocapsid core composed of protein. The nucleocapsidencloses the viral DNA and a DNA polymerase that has reversetranscriptase activity similar to retroviruses. The outer envelopecontains embedded proteins which are involved in viral binding of, andentry into, susceptible cells. The virus is one of the smallestenveloped animal viruses with a virion diameter of 42 nm, butpleomorphic forms exist, including filamentous and spherical bodies thatboth lack a core. These particles are not infectious and are composed ofthe lipid and protein that forms part of the surface of the virion,which is called the surface antigen (HBsAg), and are produced in excessduring the life cycle of the virus. The HBV virus itself is called aDane particle and consists of HBsAg, a lipid envelope, the core protein(HBcAg), the viral genome, and the Hepatitis B virus DNA polymerase. Thefunctions of the small regulatory protein (HBx) are not yet well knownbut may be related to interfering with transcription, signaltransduction, signal transduction, cell cycle progress, proteindegradation, apoptosis, or chromosomal stability. The virus alsoproduces a secreted protein called HBeAg that is an amino-terminalextension of HBcAg initiating from an upstream start codon that isinvolved in suppressing antiviral immune responses.

The genome of HBV is made of circular DNA, but it is unusual because theDNA is not fully double-stranded in the virion. One end of the fulllength strand is linked to the viral DNA polymerase. The genome is3020-3320 nucleotides long (for the full length strand) and 1700-2800nucleotides long (for the short length strand). The negative-sense,(non-coding), strand is the complete strand and it is complementary tothe viral mRNA. The viral DNA is found in the nucleus soon afterinfection of the cell. The partially double-stranded DNA is renderedfully double-stranded shortly after infection of a cell by completion ofthe (+) sense strand and removal of a protein molecule from the (−)sense strand and a short sequence of RNA from the (+) sense strand. Ashort terminal duplication of are removed from the ends of the (−)sensestrand and the ends are rejoined. The mature nuclear form of the genomeis called the “cccDNA.” The cccDNA is the template for transcription ofall of the viral mRNAs.

There are four known genes encoded by the genome called C, X, P, and S.The core protein (HBcAg) is coded for by gene C, and its start codon ispreceded by an upstream in-frame AUG start codon from which the pre-coreprotein is produced. HBeAg is produced by proteolytic processing of thepre-core protein. The DNA polymerase is encoded by gene P. Gene S is thegene that codes for the surface antigens (HBsAg). The HBsAg gene is onelong open reading frame but contains three in frame “start” (ATG) codonsthat divide the gene into three sections, pre-S1, pre-S2, and S. Becauseof the multiple start codons, polypeptides of three different sizescalled large, middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S) areproduced. The function of the protein coded for by gene X is not fullyunderstood, but it has pleiotropic regulatory functions in both thecytoplasm and nucleus.

There are at least eight known genotypes labeled A through H. A possiblenew “I” genotype has been described, but acceptance of this notation isnot universal. Different genotypes may respond to treatment in differentways. The genotypes differ by at least 8% of the sequence and havedistinct geographical distributions and this has been associated withanthropological history. Type F which diverges from the other genomes by14% is the most divergent type known. Type A is prevalent in Europe,Africa and South-east Asia, including the Philippines. Type B and C arepredominant in Asia; type D is common in the Mediterranean area, theMiddle East and India; type E is localized in sub-Saharan Africa; type F(or H) is restricted to Central and South America. Type G has been foundin France and Germany. Genotypes A, D and F are predominant in Braziland all genotypes occur in the United States with frequencies dependenton ethnicity. The E and F strains appear to have originated inaboriginal populations of Africa and the New World, respectively. Withinthese genotypes, 24 subtypes have been described which differ by 4-8% ofthe genome:

-   -   Type A has two subtypes: Aa (A1) in Africa/Asia and the        Philippines and Ae (A2) in Europe/United States.    -   Type B has two distinct geographical distributions: Bj/B1        (‘j’—Japan) and Ba/B2 (‘a’—Asia). Type Ba has been further        subdivided into four clades (B2-B4).    -   Type C has two geographically subtypes: Cs (C1) in South-east        Asia and Ce (C2) in East Asia. The C subtypes have been divided        into five clades (C1-C5). A sixth clade (C6) has been described        in the Philippines but only in one isolate to date. Type C1 is        associated with Vietnam, Myanmar and Thailand; type C2 with        Japan, Korea and China; type C3 with New Caledonia and        Polynesia; C4 with Australia; and C5 with the Philippines. A        further subtype has been described in Papua, Indonesia.    -   Type D has been divided into 7 subtypes (D1-D7).    -   Type F has been subdivided into 4 subtypes (F1-F4). F1 has been        further divided in to 1a and 1b. In Venezuela subtypes F1, F2,        and F3 are found in East and West Amerindians. Among South        Amerindians only F3 was found. Subtypes Ia, III, and IV exhibit        a restricted geographic distribution (Central America, the North        and the South of South America respectively) while clades 1b and        II are found in all the Americas except in the Northern South        America and North America respectively.

The life cycle of hepatitis B virus is complex. Hepatitis B is one of afew known non-retroviral viruses which use reverse transcription as apart of its replication process:

Attachment—

-   -   The virus gains entry into the cell by binding to a receptor on        the surface of the cell and enters it by endocytosis.

Penetration—

-   -   The virus membrane then fuses with the host cell's membrane        releasing the DNA and core proteins into the cytoplasm.

Uncoating—

-   -   Because the virus multiplies via RNA made by a host enzyme, the        viral genomic DNA has to be transferred to the cell nucleus by        host proteins. The core proteins dissociate from the partially        double-stranded viral DNA is then made fully double-stranded and        transformed into covalently closed circular DNA (cccDNA) that        serves as a template for transcription of four viral mRNAs.

Replication—

-   -   The cccDNA is the transcriptional template for all of HBV's        RNAs. The largest of the mRNAs is called the pre-core mRNA that        encodes HBeAg. A slightly shorter mRNA is called the pregenomic        RNA that encodes the HBcAg and the viral DNA polymerase. Both        the precore and pregenomic RNAs are longer than the viral        genome, but only the pregenomic RNA is packaged into nascent        core particles along with the viral polymerase. Reverse        transcription within the capsids is catalyzed by the coordinate        activity of the viral DNA polymerase's reverse transcriptase and        ribonuclease H activities and results in the partially        double-stranded viral DNA found within HBV virions.

Assembly and Release—

-   -   Progeny virions are formed budding of the viral capsid particles        containing the viral DNA into endoplasmic-reticulum-derived        membranes, where they pick up their envelope and HBsAgs are        released from the cell by non-cytolytic secretion or are        returned to the nucleus and re-cycled to produce even more        copies of the nuclear cccDNA.

2. Treatment

Currently, there are seven FDA approved drugs in the U.S. to treatchronic HBV: Intron A® (Interferon Alpha), Pegasys® (PegylatedInterferon), Epivir HBV® (Lamivudine), Hepsera® (Adefovir), Baraclude®(Entecavir), Tyzeka® (Telbivudine), and Viread® (Tenofovir).

Adefovir, previously called bis-POM PMEA, with trade names Preveon® andHepsera®, is an orally-administered nucleotide analog reversetranscriptase inhibitor (ntRTI). It can be formulated as the pivoxilprodrug adefovir dipivoxil. Adefovir works by blocking reversetranscriptase, the enzyme that is crucial for the hepatitis B virus(HBV) to reproduce in the body because it synthesizes the viral DNA. Itis approved for the treatment of chronic hepatitis B in adults withevidence of active viral replication and either evidence of persistentelevations in serum aminotransferases (primarily ALT) or histologicallyactive disease. The main benefit of adefovir over drugs like lamivudine(below) is that it takes a much longer period of time before the virusdevelops resistance to it. Adefovir dipivoxil contains twopivaloyloxymethyl units, making it a prodrug form of adefovir.

Lamivudine (2′,3′-dideoxy-3′-thiacytidine, commonly called 3TC) is apotent nucleoside analog reverse transcriptase inhibitor (nRTI). It ismarketed by GlaxoSmithKline with the brand names Zeffix®, Heptovir®,Epivir®, and Epivir-HBV®. Lamivudine has been used for treatment ofchronic hepatitis B at a lower dose than for treatment of HIV. Itimproves the seroconversion of HBeAg positive hepatitis B and alsoimproves histology staging of the liver. Long term use of lamivudineunfortunately leads to emergence of a resistant hepatitis B virusmutants with alterations to the key YMDD motif in the reversetranscriptase active site. Despite this, lamivudine is still used widelyas it is well tolerated and as it is less expensive than the newer drugsand is the only anti-HBV drug many people in emerging economies canafford.

Lamivudine is an analogue of cytidine. It can inhibit both types (1 and2) of HIV reverse transcriptase and also the reverse transcriptase ofhepatitis B. It is phosphorylated to active metabolites that compete forincorporation into viral DNA. It inhibits the HIV reverse transcriptaseenzyme competitively and acts as a chain terminator of DNA synthesis.The lack of a 3′—OH group in the incorporated nucleoside analogueprevents the formation of the 5′ to 3′ phosphodiester linkage essentialfor DNA chain elongation, and therefore, the viral DNA growth isterminated. Lamivudine is administered orally, and it is rapidlyabsorbed with a bio-availability of over 80%. Some research suggeststhat lamivudine can cross the blood-brain barrier.

Entecavir, abbreviated ETV, is an oral antiviral drug used in thetreatment of hepatitis B infection. It is marketed under the trade namesBaraclude® (BMS) and Entaliv® (DRL). Entecavir is a nucleoside analog(more specifically, a guanosine analogue) that inhibits reversetranscription and DNA replication thus preventing transcription in theviral replication process. The drug's manufacturer claims that entecaviris more efficacious than previous agents used to treat hepatitis B(lamivudine and adefovir). Entecavir was approved by the U.S.FDA inMarch 2005 and is used to treat chronic hepatitis B. It also helpsprevent the hepatitis B virus from multiplying and infecting new livercells. Entecavir is also indicated for the treatment of chronichepatitis B in adults with HIV/AIDS infection. However, entecavir is notactive against HIV.

Telbivudine is an antiviral drug used in the treatment of hepatitis Binfection. It is marketed by Swiss pharmaceutical company Novartis underthe trade names Sebivo® (Europe) and Tyzeka® (United States). Clinicaltrials have shown it to be significantly more effective than lamivudineor adefovir, and less likely to cause resistance. Telbivudine is asynthetic thymidine nucleoside analogue; it is the L-isomer ofthymidine. It is taken once daily.

Tenofovir disoproxil fumarate (TDF or PMPA), marketed by Gilead Sciencesunder the trade name Viread®, it is also a nucleotide analogue reversetranscriptase inhibitor (nRTIs) which blocks the HBV reversetranscriptase, an enzyme crucial to viral production. Tenofovirdisoproxil fumarate is a prodrug form of tenofovir. Tenofovir isindicated in combination with other antiretroviral agents for thetreatment of HIV-1 infection in adults. This indication is based onanalyses of plasma HIV-1 RNA levels and CD4 cell counts in controlledstudies of tenofovir in treatment-naive and treatment-experiencedadults. There are no study results demonstrating the effect of tenofoviron the clinical progression of HIV. It also has activity againstwild-type and lamivudine-resistant HBV.

C. NUCLEOTIDYL TRANSFERASE SUPERFAMILY ENZYMES

The inhibitors screened in this project were selected for their abilityto inhibit the HIV RNAse H and/or integrase enzymes (or to be closechemical analogs of known inhibitors). The RNAse H and integrase aremembers of the nucleotidyl transferase superfamily (NTS) whose membersshare a similar protein fold and enzymatic mechanisms (Yang 1995).Therefore, the presumed targets of the anti-herpesvirus compoundsclaimed here are viral and/or cellular NTS enzymes. RNAse H enzymes(Hostomsky et al., 1993a; 1993b; 1993c) digest RNA when it is hybridizedto DNA. Their physiological roles include removal of RNA primers duringDNA synthesis, removal of abortive transcription products, and removalof RNA strands following reverse transcription by viruses orretrotransposons. Integrase enzymes cleave DNA strands and catalyze thecovalent insertion of another DNA strand at the cleavage site.Consequently, the presumed mechanism of action for the herpesvirusinhibitors is through suppression of one or more of the nucleolytic orrecombination-related activities essential for replication of theherpesvirus DNA.

The NTS family of enzymes includes E. coli RNase H I and II (Katayanagiet al., 1990, Yang et al., 1990 and Lai et al., 2000); human RNase H 1and 2 (Lima et al., 2001, Frank et al., 1998 and Frank et al., 1998);the RuvC Holiday junction resolvase (Ariyoshi et al., 1994); and theArgonaute RNAse (Parker et al., 2004 and Song et al., 2004); retroviralRNase H enzymes including the HIV enzyme (Nowotny 2009); retroviralintegrases including the HIV integrase (Dyda et al., 1994); and thehepatitis B virus (HBV) RNase H (Tavis et al., 2013). These enzymesfunction in a wide range of nucleic acid metabolic events, including RNAand DNA digestion, DNA recombination, DNA integration, DNA excision,replication fork repair, DNA repair, miRNA maturation, andmiRNA-directed RNA cleavage. The canonical RNase H structure containsabout 100 amino acids that fold into a 5-stranded β-sheet overlaid with3 α-helices arranged like an “H”. Within the active site are fourconserved carboxylates (the “DEDD” motif) that coordinate two divalentcations (Nowotny et al., 2005).

The RNase H enzymatic mechanism is believed to involve both divalentcations (Klumpp et al., 2003; Yang and Steitz, 1995), although a 1-ionmechanism has been proposed (Goedken and Marqusee, 2001; Keck et al.,1998). There are 3 classes of RNAse Hs distinguished by how they bind totheir substrates. RNA binding by the “stand-alone” class typified by E.coli RNAse H I is promoted by a basic “handle” region (Hostomsky et al.,1993; Kwun et al., 2001). Eukaryotic RNase Hs typically contain a “RHBD”domain that influences nucleic acid binding. Finally, substrate bindingby the retroviral enzymes can either be a property of the RNase H domainitself (e.g., Moloney murine leukemia virus) or may require the reversetranscriptase domain to provide sufficient affinity for the nucleic acidsubstrate (e.g., HIV) (Hostomsky et al., 1993; Smith et al., 1994).

The HBV RNase H is a NTS enzyme. Mutational analysis of the HBV RNase Hrevealed the DEDD active site residues to be D702, E731, D750, and D790(numbering for HBV strain adw2) (Gerelsaikhan et al., 1996; Tavis etal., 2013). Data obtained with the HBV RNase H will be used as anexample to establish how anti-RNase H drug discovery can be conductedand to establish utility of the claimed compounds against HBVreplication.

HIV reverse transcription requires a virally encoded RNase H activity toremove the viral RNA after it has been copied into DNA (Freed andMartin, 2007). Consequently, the HIV RNase H activity has attracted muchattention as a drug target (Billamboz et al., 2011; Bokesch et al.,2008; Budihas et al., 2005; Chung et al., 2011; Chung et al., 2010; Diet al., 2010; Didierjean et al., 2005; Fuji et al., 2009; Himmel et al.,2009; Himmel et al., 2006; Kirschberg et al., 2009; Klarmann et al.,2002; Klumpp et al., 2003; Klumpp and Mirzadegan, 2006; Shaw-Reid etal., 2003; Su et al., 2010; Takada et al., 2007; Wendeler et al., 2008;Williams et al., 2010). Over 100 anti-HIV RNase H compounds, based on awide variety of chemical scaffolds, have been reported (Chung et al.,2011; Klumpp and Mirzadegan, 2006). They typically have inhibitoryconcentration-50% (IC₅₀) values in the low μM range. The large majorityof these compounds inhibit the RNase H by chelating divalent cations inthe active site (Billamboz et al., 2011; Chung et al., 2011; Fuji etal., 2009; Himmel et al., 2009; Kirschberg et al., 2009; Su et al.,2010), but compounds that alter the enzyme's conformation or itsinteraction with nucleic acids have also been reported (Himmel et al.,2006; Wendeler et al., 2008). The inhibitors typically have EC₅₀ values˜10× higher than the IC₅₀ values, and they often cause modestcytotoxicity, leading to therapeutic indexes (TI) that are usually <10.Second-generation inhibitors with substantially improved efficacy havebeen reported, (Billamboz et al., 2011; Chung et al., 2011; Williams etal., 2010), and compounds with efficacy and TI values appropriate for ahuman drug exist (Himmel et al., 2006; Williams et al., 2010).

None of the anti-HIV RNase H compounds have entered clinical trials yet.This is due in part to their relatively low TI values but also to thelarge number of approved and developmental anti-HIV drugs, raisingdoubts about the marketability of anti-HIV RNase H compounds. Despitethese challenges, the HIV RNase H remains a target of intensive ongoingdrug development, as is evidenced by the large number of groups workingin the field (Billamboz et al., 2011; Bokesch et al., 2008; Budihas etal., 2005; Chung et al., 2011; Chung et al., 2010; Di et al., 2010;Didierjean et al., 2005; Fuji et al., 2009; Himmel et al., 2009; Himmelet al., 2006; Kirschberg et al., 2009; Klarmann et al., 2002; Klumpp etal., 2003; Klumpp and Mirzadegan, 2006; Shaw-Reid et al., 2003; Su etal., 2010; Takada et al., 2007; Wendeler et al., 2008; Williams et al.,2010).

Because both the RNase H and integrase are NTS enzymes, some anti-RNaseH compounds can inhibit the HIV integrase, and some anti-integrasecompounds can inhibit the RNase H (Klarmann et al., 2002, Williams etal., 2010 and Billamboz et al., 2011). Despite this cross-inhibitorypotential, resistance mutations to HIV DNA polymerase or integrase drugshave not led to cross-resistance to RNase H inhibitors (Billamboz etal., 2011 and Himmel et al., 2006).

HBV reverse transcription requires two viral enzymatic activities thatare both located on the viral reverse transcriptase protein. The DNApolymerase activity synthesizes new DNA and is targeted by thenucleos(t)ide analogs. The RNase H destroys the viral RNA after it hasbeen copied into DNA. Inhibiting the RNAse H would block DNA synthesisand consequently halt viral replication, but anti-HBV RNase H drugs havenot been developed because enzyme suitable for drug screening could notbe readily made. One of the inventors recently produced activerecombinant HBV RNase H and identified 35 inhibitors of the RNase H(Table 1; Tavis et al., 2013; Hu et al., 2013; Tavis and Lomonosova,2015; Lu, et al., 2015; and Cai, et al., 2014).

These examples of cross-inhibition of NTS enzymes by RNase H andintegrase inhibitors provide the precedent upon which the studies withthe herpesviruses rest.

D. CHEMICAL ENTITY

The compound of the present disclosure appears to inhibit a differentenzymatic activity than the existing anti-hepatitis or anti-herpesvirusdrugs, and does so with a striking capacity to suppress virusreplication at very low toxicity to uninfected cells. This implies thatit will be effective against viral isolates resistant to the existingdrugs and suggests that these drugs could be combined effectively withthe existing drugs to both increase efficacy and to reduce the rate ofresistance development to either drug. Furthermore, the compound wasmore effective against HSV-2 than a currently accepted first linetherapy, acyclovir, indicating that it may be more effective than theexisting drugs when formulated for pharmaceutical delivery. In otherembodiments, the present compounds may be used to treat infections ofhepatitis B virus or used in combination with other hepatitis B virustherapies.

The compound of the present disclosure is represented by the formulabelow:

wherein:

-   -   R₁ is aryl_((C≤12)), aralkyl_((C≤18)), heteroaryl_((C≤12)),        alkylamino_((C≤12)), dialkylamino_((C≤12)), arylamino_((C≤12)),        diarylamino_((C≤12)), aralkylamino_((C≤18)),        diaralkylamino_((C≤18)), or a substituted version of any of        these groups;    -   R₂ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and    -   R₃ is hydrogen, amino, carboxyl, cyano, halo, hydroxy, nitro,        hydroxysulfonyl, or sulfonylamine; or alkyl_((C≤8)),        aryl_((C≤8)), acyl_((C≤8)), alkoxy_((C≤8)), acyloxy_((C≤8)),        amido_((C≤8)), or substituted version of any of these groups;        -   X₂ is hydrogen or —C(O)R_(a), wherein: R_(a) is hydroxy,            alkoxy_((C≤8)), or substituted alkoxy_((C≤8)); or

a compound of the formula:

wherein:

-   -   R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₅ and R₈ are each independently hydrogen, alkyl_((C≤8)), or        substituted alkyl_((C≤8));    -   R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substituted        alkyl_((C≤8)); and    -   R₇ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), or a        substituted version of any of these groups;

a compound of the formula:

wherein:

-   -   R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),        heteroaryl_((C≤12)), or a substituted version of any of these        groups;    -   R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));        and    -   R₁₁ is hydrogen or Y₁—O—X₁—OR₁₂; wherein:        -   Y₁ is alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8));        -   X₁ is arenediyl_((C≤12)), heteroarenediyl_((C≤12)), or a            substituted version of either of these groups;        -   R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)),            or a substituted version of any of these groups; or

or a pharmaceutically acceptable salt or tautomer thereof.

TABLE 1 Non-Limiting Examples of Compounds Compound ID Structure #41

#49

#150

#151

#191

#208

#210

#211

The compound of the disclosure contains one or moreasymmetrically-substituted carbon or nitrogen atoms, and may be isolatedin optically active or racemic form. Thus, all chiral, diastereomeric,racemic form, epimeric form, and all geometric isomeric forms of thechemical formula are intended, unless the specific stereochemistry orisomeric form is specifically indicated. The compound may occur as aracemate and racemic mixtures, single enantiomers, diastereomericmixtures and individual diastereomers. In some embodiments, a singleenantiomer or diastereomer is obtained. The chiral centers of thecompound of the present disclosure can have the S or the Rconfiguration.

Chemical formulas used to represent the compound of the disclosure willtypically only show one of possibly several different tautomers. Forexample, many types of ketone groups are known to exist in equilibriumwith corresponding enol groups. Regardless of which tautomer is depictedfor a given compound, and regardless of which one is most prevalent, alltautomers of a given chemical formula are intended.

The compound of the disclosure may also have the advantage of being moreefficacious than, be less toxic than, be longer acting than, be morepotent than, produce fewer side effects than, be more easily absorbedthan, and/or have a better pharmacokinetic profile (e.g., higher oralbioavailability and/or lower clearance) than, and/or have other usefulpharmacological, physical, or chemical properties over, compounds knownin the prior art, whether for use in the indications stated herein orotherwise.

In addition, atoms making up the compound of the present disclosure areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present disclosure may be replacedby a silicon atom(s). Furthermore, it is contemplated that one or moreoxygen atom(s) of a compound of the present disclosure may be replacedby a sulfur or selenium atom(s).

The compound of the present disclosure may also exist in prodrug form.Since prodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing,etc.), the compound employed in some methods of the disclosure may, ifdesired, be delivered in prodrug form. Thus, the disclosure contemplatesprodrugs of the compound of the present disclosure as well as methods ofdelivering prodrugs. Prodrugs of the compound employed in the disclosuremay be prepared by modifying functional groups present in the compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound. Accordingly, prodrugsinclude, for example, compounds described herein in which a hydroxygroup is bonded to any group that, when the prodrug is administered to asubject, cleaves to form a hydroxy group.

It should be recognized that the particular anion or cation forming apart of any salt of this disclosure is not critical, so long as thesalt, as a whole, is pharmacologically acceptable. Additional examplesof pharmaceutically acceptable salts and their methods of preparationand use are presented in Handbook of Pharmaceutical Salts: Properties,and Use (2002), which is incorporated herein by reference.

2. Definitions

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “nitro” means —NO₂; imino means ═NH; “cyano” means —CN;“isocyanate” means —N═C═O; “azido” means —N₃; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof; “mercapto” means —SH; and “thio” means ═S; “sulfonyl” means—S(O)₂—; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond, and “≡” means triple bond. The symbol “----”represents an optional bond, which if present is either single ordouble. The symbol “

” represents single bond or a double bond. Thus, the formula

covers, for example,

And it is understood that no one such ring atom forms part of more thanone double bond. Furthermore, it is noted that the covalent bond symbol“—”, when connecting one or two stereogenic atoms, does not indicate anypreferred stereochemistry. Instead, it covers all stereoisomers as wellas mixtures thereof. The symbol “

”, when drawn perpendicularly across a bond (e.g.,

for methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this mannerfor larger groups in order to assist the reader in unambiguouslyidentifying a point of attachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the geometry around a double bond (e.g.,either E or Z) is undefined. Both options, as well as combinationsthereof are therefore intended. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to that atom. A bold dot on a carbon atom indicates that thehydrogen attached to that carbon is oriented out of the plane of thepaper.

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the chemical groups and compound classes, the number of carbon atomsin the group or class is as indicated as follows: “Cn” defines the exactnumber (n) of carbon atoms in the group/class. “C≤n” defines the maximumnumber (n) of carbon atoms that can be in the group/class, with theminimum number as small as possible for the group/class in question,e.g., it is understood that the minimum number of carbon atoms in thegroup “alkenyl_((C≤8))” or the class “alkene_((C≤8))” is two. Comparewith “alkoxy_((C≤10))”, which designates alkoxy groups having from 1 to10 carbon atoms. “Cn−n′” defines both the minimum (n) and maximum number(n′) of carbon atoms in the group. Thus, “alkyl_((C2-10))” designatesthose alkyl groups having from 2 to 10 carbon atoms. These carbon numberindicators may precede or follow the chemical groups or class itmodifies and it may or may not be enclosed in parenthesis, withoutsignifying any change in meaning. Thus, the terms “C5 olefin”,“C5-olefin”, “olefin_((C5))”, and “olefin_(C5)” are all synonymous. Whenany of the chemical groups or compound classes defined herein ismodified by the term “substituted”, any carbon atom(s) in a moietyreplacing a hydrogen atom is not counted. Thus methoxyhexyl, which has atotal of seven carbon atoms, is an example of a substitutedalkyl_((C1-6)).

The term “saturated” when used to modify a compound or chemical groupmeans the compound or chemical group has no carbon-carbon double and nocarbon-carbon triple bonds, except as noted below. When the term is usedto modify an atom, it means that the atom is not part of any double ortriple bond. In the case of substituted versions of saturated groups,one or more carbon oxygen double bond or a carbon nitrogen double bondmay be present. And when such a bond is present, then carbon-carbondouble bonds that may occur as part of keto-enol tautomerism orimine/enamine tautomerism are not precluded. When the term “saturated”is used to modify a solution of a substance, it means that no more ofthat substance can dissolve in that solution.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound or chemical group so modified is an acyclicor cyclic, but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by singlecarbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or morecarbon-carbon double bonds (alkenes/alkenyl) or with one or morecarbon-carbon triple bonds (alkynes/alkynyl).

The term “aromatic” when used to modify a compound or a chemical grouprefers to a planar unsaturated ring of atoms with 4n+2 electrons in afully conjugated cyclic π system.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched acyclic structure, and no atomsother than carbon and hydrogen. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂(i-Pr, ^(i)Pr or isopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl),—CH₂CH(CH₃)₂(isobutyl), —C(CH₃)₃(tert-butyl, t-butyl, t-Bu or ^(t)Bu),and —CH₂C(CH₃)₃(neo-pentyl) are non-limiting examples of alkyl groups.The term “alkanediyl” when used without the “substituted” modifierrefers to a divalent saturated aliphatic group, with one or twosaturated carbon atom(s) as the point(s) of attachment, a linear orbranched acyclic structure, no carbon-carbon double or triple bonds, andno atoms other than carbon and hydrogen. The groups —CH₂— (methylene),—CH₂CH₂—, —CH₂C(CH₃)₂CH₂—, and —CH₂CH₂CH₂— are non-limiting examples ofalkanediyl groups. The term “alkylidene” when used without the“substituted” modifier refers to the divalent group ═CRR′ in which R andR′ are independently hydrogen or alkyl. Non-limiting examples ofalkylidene groups include: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. An “alkane”refers to the class of compounds having the formula H—R, wherein R isalkyl as this term is defined above. When any of these terms is usedwith the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂C1, —CF₃, —CH₂CN, —CH₂C(O)OH,—CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂,—CH₂N(CH₃)₂, and —CH₂CH₂C1.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, anda monovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic groupwith two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl, aryl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. If more than one ring is present, the rings may be fused orunfused. Unfused rings may be connected via one or more of thefollowing: a covalent bond, alkanediyl, or alkenediyl groups (carbonnumber limitation permitting). Non-limiting examples of arenediyl groupsinclude:

An “arene” refers to the class of compounds having the formula H—R,wherein R is aryl as that term is defined above. Benzene and toluene arenon-limiting examples of arenes. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and2-phenyl-ethyl. When the term aralkyl is used with the “substituted”modifier one or more hydrogen atom from the alkanediyl and/or the arylgroup has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than onering is present, the rings may be fused or unfused. As used herein, theterm does not preclude the presence of one or more alkyl, aryl, and/oraralkyl groups (carbon number limitation permitting) attached to thearomatic ring or aromatic ring system. Non-limiting examples ofheteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im),isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl(pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl,quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl.The term “N-heteroaryl” refers to a heteroaryl group with a nitrogenatom as the point of attachment. A “heteroarene” refers to the class ofcompounds having the formula H—R, wherein R is heteroaryl. Pyridine andquinoline are non-limiting examples of heteroarenes. When these termsare used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or arylas those terms are defined above. The groups, —CHO, —C(O)CH₃ (acetyl,Ac), —C(O)CH₂CH₃, —C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, and—C(O)C₆H₄CH₃ are non-limiting examples of acyl groups. A “thioacyl” isdefined in an analogous manner, except that the oxygen atom of the group—C(O)R has been replaced with a sulfur atom, —C(S)R. The term “aldehyde”corresponds to an alkyl group, as defined above, attached to a —CHOgroup. When any of these terms are used with the “substituted” modifierone or more hydrogen atom (including a hydrogen atom directly attachedto the carbon atom of the carbonyl or thiocarbonyl group, if any) hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl),—CO₂CH₃ (methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and—CON(CH₃)₂, are non-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples include: —OCH₃ (methoxy), —OCH₂CH₃ (ethoxy),—OCH₂CH₂CH₃, —OCH(CH₃)₂(isopropoxy), —OC(CH₃)₃(tert-butoxy), —OCH(CH₂)₂,—O-cyclopentyl, and —O-cyclohexyl. The terms “cycloalkoxy”,“alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as —OR, in which R is cycloalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl,respectively. The term “alkylthio” and “acylthio” when used without the“substituted” modifier refers to the group —SR, in which R is an alkyland acyl, respectively. The term “alcohol” corresponds to an alkane, asdefined above, wherein at least one of the hydrogen atoms has beenreplaced with a hydroxy group. The term “ether” corresponds to analkane, as defined above, wherein at least one of the hydrogen atoms hasbeen replaced with an alkoxy group. When any of these terms is used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples include: —NHCH₃ and —NHCH₂CH₃. Theterm “dialkylamino” when used without the “substituted” modifier refersto the group —NRR′, in which R and R′ can be the same or different alkylgroups, or R and R′ can be taken together to represent an alkanediyl.Non-limiting examples of dialkylamino groups include: —N(CH₃)₂ and—N(CH₃)(CH₂CH₃). The terms “cycloalkylamino”, “alkenylamino”,“alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”,“heterocycloalkylamino”, “alkoxyamino”, and “alkylsulfonylamino” whenused without the “substituted” modifier, refers to groups, defined as—NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl,heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. Anon-limiting example of an arylamino group is —NHC₆H₅. The term “amido”(acylamino), when used without the “substituted” modifier, refers to thegroup —NHR, in which R is acyl, as that term is defined above. Anon-limiting example of an amido group is —NHC(O)CH₃. The term“alkylimino” when used without the “substituted” modifier refers to thedivalent group ═NR, in which R is an alkyl, as that term is definedabove. When any of these terms is used with the “substituted” modifierone or more hydrogen atom attached to a carbon atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects, or +/−5% of the stated value.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingvertebrate organism, such as a human, monkey, cow, sheep, goat, dog,cat, mouse, rat, guinea pig, bird, fish or transgenic species thereof.In certain embodiments, the patient or subject is a primate.Non-limiting examples of human subjects are adults, juveniles, infantsand fetuses.

As generally used herein, “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of the compound of thepresent disclosure which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this disclosure is not critical, so longas the salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (2002).

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease, including reactivation.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present disclosure. The prodrugitself may or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2^(n), where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diasteromers can be resolved or separated usingtechniques known in the art. It is contemplated that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≤15%, morepreferably ≤10%, even more preferably ≤5%, or most preferably ≤1% ofanother stereoisomer(s).

“Effective amount,” “therapeutically effective amount” or“pharmaceutically effective amount” means that amount which, whenadministered to a subject or patient for treating a disease, issufficient to effect such treatment for the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease. In some embodiments, treatment of apatient afflicted with one of the pathological conditions describedherein comprises administering to such a patient an amount of compounddescribed herein which is therapeutically effective in controlling thecondition or in prolonging the survivability of the patient beyond thatexpected in the absence of such treatment. As used herein, the term“inhibition” of the condition also refers to slowing, interrupting,arresting or stopping the condition and does not necessarily indicate atotal elimination of the condition. It is believed that prolonging thesurvivability of a patient, beyond being a significant advantageouseffect in and of itself, also indicates that the condition isbeneficially controlled to some extent.

The above definitions supersede any conflicting definition in anyreference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the disclosure in terms such thatone of ordinary skill can appreciate the scope and practice the presentdisclosure.

E. THERAPEUTIC METHODS

1. Pharmaceutical Formulations

In particular embodiments, where clinical application of an activeingredient is undertaken, it will be necessary to prepare apharmaceutical composition appropriate for the intended application.Generally, this will entail preparing a pharmaceutical composition thatis essentially free of pyrogens, as well as any other impurities orcontaminants that could be harmful to humans or animals. One also willgenerally desire to employ appropriate buffers to render the complexstable and allow for uptake by target cells.

Aqueous compositions of the present disclosure comprise an effectiveamount of the active compound, as discussed above, further dispersed inpharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to compositions that do not producean adverse, allergic or other untoward reaction when administered to ananimal, or a human, as appropriate, as well as the requisite sterilityfor in vivo uses.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

Solutions of therapeutic compositions can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The therapeutic compositions of the present disclosure areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified. A typicalcomposition for such purpose comprises a pharmaceutically acceptablecarrier. For instance, the composition may contain 10 mg, 25 mg, 50 mgor up to about 100 mg of human serum albumin per milliliter of phosphatebuffered saline. Other pharmaceutically acceptable carriers includeaqueous solutions, non-toxic excipients, including salts, preservatives,buffers and the like.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate.Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto well-known parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, a controlled release patch,salve or spray. In some embodiments, the topical formulation by used foradministration to the skin, to mucosa membranes such as the eye, eyelids, the genitals, the anus, or the inside of the mouth or nose, and inparticular to the cornea.

An effective amount of the therapeutic composition is determined basedon the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection desired.

Precise amounts of the therapeutic composition also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting dose include physical and clinical state of thepatient, the route of administration, the intended goal of treatment andthe potency, stability and toxicity of the particular therapeuticsubstance.

2. Routes of Administration

Formulations of the present disclosure are suitable for oraladministration. However, the therapeutic compositions of the presentdisclosure may be administered via any common route so long as thetarget tissue is available via that route. This includes nasal, buccal,corneal, ocularly, rectal, vaginal, or topical administration, andintradermal, subcutaneous, intramuscular, intraperitoneal or intravenousinjection. As such, compositions would be formulated pharmaceutically inroute-acceptable compositions that include physiologically acceptablecarriers, buffers or other excipients.

As with dosing amounts, the timing of delivery (including intervals andtotal number of doses) depends on the judgment of the practitioner andare peculiar to each individual. Factors affecting dose include physicaland clinical state of the patient, the route of administration, theintended goal of treatment and the potency, stability and toxicity ofthe particular therapeutic substance.

3. Combination Therapy

In many clinical situations, it is advisable to use a combination ofdistinct therapies. Thus, it is envisioned that, in addition to thetherapies described above, one would also wish to provide to the patientmore “traditional” pharmaceutical hepatitis or herpesvirus therapies.Examples of standard therapies are described above. Combinations may beachieved by administering a single composition or pharmacologicalformulation that includes both agents, or with two distinct compositionsor formulations, at the same time, wherein one composition includes theagents of the present disclosure and the other includes the standardtherapy. Alternatively, standard therapy may precede or follow thepresent agent treatment by intervals ranging from minutes to weeks tomonths. In embodiments where the treatments are applied separately, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agents would still beable to exert an advantageously combined effect on the subject. In suchinstances, it is contemplated that one would administer both modalitieswithin about 12-24 hours of each other and, more preferably, withinabout 6-12 hours of each other, with a delay time of only about 12 hoursbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

It also is conceivable that more than one administration of either theagent of the present disclosure, or the standard therapy will bedesired. Various combinations may be employed, where the presentdisclosure compound is “A” and the standard therapy is “B,” asexemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated as well. Drugs suitable for suchcombinations are described above and include, but are not limited to,DNA polymerase inhibitors (nucleoside analogs), including acyclovir,famciclovir, valaciclovir, penciclovir, and ganciclovir. Additionally,it is contemplated that other antiviral agents such as a pegylatedinterferon, interferon alfa-2b, lamivudine, adefovir, telbivudine,entercavir, or tenofovir may be used in combination with the compoundsdescribed herein.

E. EXAMPLES

The following examples are included to further illustrate variousaspects of the disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques and/or compositions discovered by the inventors tofunction well in the practice of the disclosure, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the disclosure.

1. Materials and Methods

Cells and Viruses.

Vero cells were maintained in Dulbecco's modified Eagle's medium (DMEM)containing 3% newborn calf serum, 3% bovine growth serum, 2 mML-glutamine and 100 IU/mL penicillin and 0.1 mg/mL streptomycin (P/S).HSV-1 #6 and HSV-2 #1 are de-identified clinical isolates from the SaintLouis University Hospital. Stocks were prepared after a single passagein cell culture and were titered by standard plaque assay (Knipe andSpang, 1982). Wild-type HSV-2 used in FIG. 3 was laboratory strain 333.The TK-deficient mutant of HSV-2 strain 333, ΔTK-, contains a 180-bpKpnI-KpnI deletion in the UL23 open reading frame that abrogates TKactivity (McDermott, et al, 1984). ΔTK—was the generous gift of JimSmiley. Virus stocks were grown and titered on Vero cells (Morrison andKnipe, 1996).

Compound Selection Strategy.

The compound was selected for evaluation of its ability to inhibit HSVreplication based on one or more of the following criteria:

-   -   close chemical relatives of compounds that inhibit the HIV RNase        H, the HIV integrase, or the HBV RNase H    -   availability from commercial sources and/or through the NCI        compound repository.

Anti-HSV-1 and -HSV-2 Replication Assay.

Compounds to be screened were diluted in PBS supplemented to contain 2%newborn calf serum and 1% glutamine, and added in 100 μL volume toconfluent cell monolayers in 24-well cluster plates. Immediatelythereafter HSV-1 and HSV-2, diluted in the supplemented PBS medium, wereadded to the wells in 50 μl volume such that the final concentration ofcompound was 50 μM, 5 μM and 1 μM and the multiplicity of infection was0.1. The plates were incubated at 37° C. for 1 hour and thenvirus-containing inoculum was removed and the wells were washed once inPBS.

Compound, diluted to 50 μM, 5 μM and 1 μM in DMEM supplemented tocontain 2% newborn calf serum and 1% each penicillin/streptomycin, wasadded at 0.5 mL/well. Plates were incubated at 37° C. an additional 23hours, and then the plates were visually inspected through a phasecontrast microscope for cytopathic effect, and for toxic effect. Theentire contents of each well were collected by scraping. Samples werefrozen at −80° C., and then subsequently thawed, sonicated, andinfectious virus titer was determined by standard plaque assay on Verocell monolayers. Because the compounds were dissolved at 10 mM in 100%DMSO, equivalent dilutions of DMSO were added to additional wells as acontrol for effects of the diluent. Each experiment was repeated once.EC₅₀ values were determined as above except that serial dilutions of thecompound to be tested were prepared starting at 50 μM. The inhibitoryvalues were calculated by non-linear curve-fitting in GraphPad Prism.

Toxicity Assays.

Qualitative assessments of cytotoxicity were done visually by inspectingthe cells in the primary screening assays. For the quantitative assays,cells were plated in 96-well plates at 1.0×10⁴ per well. The next daythe compounds were added at 0.78 to 100 μM in a final concentration of1% (v/v) DMSO, and the cells were incubated for 24 hours underconditions identical to those employed for the viral replicationinhibition assays. Mitochondrial toxicity was measured by incubating thecells with 0.25 mg/mL thiazolyl blue tetrazolium bromide (MTT,SigmaAldrich Chemical Co.), the cultures were incubated for 60 min,metabolites were solubilized in acidic isopropanol, and absorbance wasread at 570 nm. CC₅₀ values were calculated by non-linear curve fittingusing GraphPad Prism.

Mouse Liver Microsome Assays.

Microsomal incubations are run at a final P450 concentration of 0.25 μM.The microsomal mixture is composed of 0.1M potassium phosphatecontaining 3.3 mM MgCl and 1 μM compound (final concentration). Themixture is prewarmed for 5 min at 37° C. To initiate the reaction a 12mM stock of NAPDH (1.2 mM, final concentration) is added to the warmedmicrosomal mixture and aliquots are removed at 0, 5, 10, 20 and 30 min.Samples are quenched with ice cold acetonitrile containing internalstandard and either 1.8 mM EDTA or EGTA (final concentration). Samplesare vortexed and spun down at 3200 rpm for 5 min. The supernant istransferred to 96-deep well plates and run on LC/MS/MS.

RNaseH Expression and Purification.

Recombinant HBV RNaseH and human RNaseH1 were expressed in E. coli andpartially purified by nickel-affinity chromatography as previouslydescribed in Tavis, et al. (2013). The enriched extracts were dialyzedinto 50 mM HEPES pH 7.3, 300 mM NaCl, 20% glycerol, and 5 mM DTT andstored in liquid nitrogen.

Oligonucleotide-Directed RNA Cleavage Assay.

RNaseH activity was measured using an oligonucleotide-directed RNAcleavage assay as previously described in Hu, et al., Tavis, et al., andCai, et al. (2013; 2013; and 2014, respectfully). Briefly, 6 μL proteinextract was mixed on ice with an internally ³²P-labeled 264 nt RNAderived from the Duck Hepatitis B Virus genome (DRF+ RNA) plus 3 μgoligonucleotide D2507- or its inverse-complement oligonucleotide D2526+as a negative control. This mixture was incubated with test compounds in50 mM Tris pH 8.0, 190 mM NaCl, 5 mM MgCl₂, 3.5 mM DTT, 0.05% NP40, 6%glycerol, and 1% DMSO at 42° C. for 90 minutes. Cleavage products wereresolved by denaturing polyacrylamide gel electrophoresis, detected byautoradiography, and quantified using ImageJ. Non-specific backgroundvalues were determined from the incorrect oligonucleotide negativecontrol lane and subtracted from all experimental values. IC₅₀ valueswere then calculated with GraphPad Prism using three-parameterlog(inhibitor) vs. response non-linear curve fitting with the curveminimum set to zero to reflect background subtraction.

HBV Replication Assay.

Inhibition of HBV replication was measured in HepDES19 cells aspreviously described in Cai, et al. (2014). Cells were seeded into6-well plates and incubated in DMEM/F12, 10% fetal bovine serum (FBS),1% penicillin/streptomycin (P/S) with 1 μg/mL tetracycline. Tetracyclinewas withdrawn after 24 hours. The test compound was applied to duplicatewells 48 hours later in medium containing a final DMSO concentration of1%, and medium containing the compound was refreshed daily for thefollowing two days. Cells were harvested and non-encapsidated nucleicacids were digested with micrococcal nuclease (New England Biolabs). HBVDNA was purified from capsids using QIAamp Cador Pathogen Mini Kit(Qiagen) with proteinase K incubation overnight at 37° C. TaqMan PCR wasperformed for 40 cycles at an annealing temperature of 60° C. Primersand probe (IDT Inc.) for the plus-polarity strand were:5′CATGAACAAGAGATGATTAGGCAGAG3′ (SEQ ID NO: 1);5′GGAGGCTGTAGGCATAAATTGG3′ (SEQ ID NO: 2);5′/56-FAM/CTGCGCACC/ZEN/AGCACCATGCA/3IABkFQ (SEQ ID NO: 3). Primers andprobe for the minus-polarity strand were: 5′GCAGATGAGAAGGCACAGA3′ (SEQID NO: 4; 5′CTTCTCCGTCTGCCGTT3′ (SEQ ID NO: 5);5′/56-FAM/AGTCCGCGT/ZEN/AAAGAGAGGTGCG/3IABkFQ (SEQ ID NO: 6).

MTT Cytotoxicity Assays.

1.0×10⁴ HepDES19 cells per well were seeded in 96-well plates andincubated in DMEM with 10% FBS plus 1% P/S, 1% non-essential aminoacids, and 1% glutamine. Compounds were diluted in medium to theindicated concentrations plus 1% DMSO and added to cells 24 hours afterplating, with each concentration tested in triplicate. Medium containingthe compound was refreshed daily for the next two days. Thiazolyl bluetetrazolium bromide (MTT, Sigma-Aldrich) was added to 0.25 mg/mL, thecultures were incubated for 60 minutes, metabolites were solubilized inacidic isopropanol, and absorbance was read at 570 nM.

2. Results

Compound Selection Strategy.

This compound was selected based on its structural similarity topolyoxygenated heterocycle compounds with anti-microbial activity.Piroctone olamine (octopirox; #191) is an approved antifungal in Europe.Its structure is shown in FIG. 1. Nine napthyridinones were tested forcapacity to inhibit the nuclease activity of pUL15C (Masaoka et al.,2016). Seven of these inhibited nuclease activity with IC₅₀ values ≤2μM, and two of the seven compounds (#151 and 155) strongly suppressedHSV-1 and HSV-2 replication in cell culture at 5 μM or less (Table 2).

Primary Screening for Inhibition of HSV-1 and HSV-2 Replication.

Efficacy of this compound against both HSV-1 and HSV-2 was initiallyassessed at 50, 5 and 1 μM in a semi-quantitative replication inhibitionassay. Inhibition was categorized as negligible (<1 log₁₀ at 50 μMrelative to the DMSO-treated control), intermediate (1 to 3 log₁₀suppression, which equals 10- to 1,000-fold reduction), or strong (3 to6 log₁₀ suppression, or 1,000- to 1,000,000-fold reduction). Compound#191 (piroctone olamine or octopirox) showed strong inhibitory activityat 50 and 5 μM against HSV-1 and HSV-2 (Table 2). Compound #191maintained intermediate inhibition of HSV-1 and HSV-2 even at 1 μM(Table 2). Thus, it inhibited HSV-1 by 3.61 log₁₀ (4,074-fold) and HSV-2by 4.59 log₁₀ (39,000-fold) at 5 μM. For comparison, the approvedanti-HSV drug acyclovir inhibited HSV-1 replication in this assay by4.22 log₁₀ (16,600-fold) and HSV-2 by only 3.6 log₁₀ (3,980-fold) at 5μM. Therefore, this polyoxygenated heterocycle compound inhibits bothHSV-1 and HSV-2 replication as well as or better than acyclovir.

TABLE 2 Compound HSV-1 HSV-2 Anti- Number and Log₁₀ suppression Log₁₀suppression human Reference 50 μM 5 μM 1 μM EC₅₀ 50 μM 5 μM 1 μM EC₅₀RNase H1 CC₅₀ #191 (Piroctone 3.85 3.53 2.71 1.57 μM 4.25 4.53 1.70 1.53μM — >100 olamine) #208 2.63 0.15 0.26 0.11 #211 1.6 0.08 1.81 0.04Acyclovir 5.39 3.61 nd 0.16 μM 5.25 3.91 nd 1.44 μM nd >100 nd, notdetermined.

Compound Toxicity.

Visual inspection of the infected cells at the end of the 24 hourinfection window through a phase-contrast microscope revealedsubstantially less cytopathic effect (CPE) than the DMSO-treated controlwells without appearance of apoptosis or necrosis.

A quantitative toxicity measurement was conducted. Toxicity was assessedby measuring release of intracellular proteases into the culture mediumdue to cellular lysis. Cells were plated in 96-well plates at 1.0×10⁴per well. The next day compound was added in concentrations ranging from0.78 to 100 μM in a final concentration of 1% (v/v) DMSO. The cells wereincubated for 24 hours under conditions identical to those employed forthe viral replication inhibition assays, and then mitochondrial activitywas measured with the MTT assay (Sigma Aldrich) according to themanufacturer's instructions. Percent viability was determined for eachcompound concentration from the luminosity data, and then 50% cytotoxicconcentration (CC₅₀) value was calculated by non-linear curve fittingusing GraphPad Prism. Consistent with the subjective assessments oftoxicity, #191 had a CC₅₀ value >100 μM under these conditions (Table1). #191 did not inhibit the activity of human RNaseH1 (Table 1),another indicator of low toxicity.

Quantitative HSV-1 and HSV-2 Replication Inhibition Assays.

To obtain a more quantitative evaluation of the inhibitory potential ofcompound #191, EC₅₀ values were determined against HSV-1 and HSV-2. ForHSV-1, the EC₅₀ value was 0.27 μM. For HSV-2, the value was 0.60 μM. Forcomparison, acyclovir had an EC₅₀ of 0.16 and 1.44 μM versus HSV-1 andHSV-2, respectively (Table 1). Overall, these quantitative dataconfirmed the highly effective inhibition of HSV-1 and HSV-2 by compound#191 in the semi-quantitative assay. They also reinforce the observationthat this compound can efficiently inhibit both HSV-1 and HSV-2, andthey strengthen the conclusion that the strong inhibitor identified herehas equivalent or superior activity against the herpes simplex virusesthan the approved drug acyclovir.

Compound #191 suppressed replication of three primary clinical isolatesof HSV-2 by >4.7 log₁₀ at 5 μM (FIG. 2), indicating that it is activeagainst a range of clinically relevant virus strains.

Inhibition of an Acyclovir-Resistant HSV-2 Mutant.

ACV is a nucleoside analog prodrug that must be phosphorylated by theviral thymidine kinase (TK) for it to become a substrate for the viralDNA polymerase (Elion, et al., 1977). HSV TK-deficient mutants aretherefore insensitive to ACV. Because viral resistance to ACV and othernucleoside analogs is a significant medical problem (Field and Biron,1994; Coen, 1991; Wang, et al, 2011; Duan, et al., 2009; Duan, et al.,2008; Pelosi, et al., 1992), especially in immunocompromised patients(Reyes, et al., 2013; Levin, et al., 2004; Gilbert, et al., 2002; Schmitand Boivin, 1999), the inventors considered whether defined TK-deficientmutants of HSV-2 would be sensitive to other compounds such as compound#191. Vero cells were infected with a laboratory strain of HSV-2 and anengineered TK-deficient mutant of the same strain. The cells weretreated with 5 μM ACV or compound #191 as was done in the primaryscreening assays, and viral yields 24 hours post-infection were measuredby plaque assay. ACV 5 μM inhibited wild-type HSV-2 replication100-fold, but it had little effect on the TK-mutant (FIG. 3). In markedcontrast, compound #191 efficiently inhibited the wild-type HSV-2 andthe TK-mutant strain. Therefore, this compound does not requirephosphorylation by the viral TK gene to be active, confirming thatcompound #191 suppress HSV-2 replication in a different manner than ACV.These data also demonstrate that compound #191 are stronger inhibitorsof HSV-2 than ACV at 5 μM.

Therapeutic Implications.

The high suppressive activities against the herpesviruses observed withthis compound (as much as 4.59 log₁₀ at 5 μM) and its minimal short-termtoxicity implies that it may be suitable for use as an anti-viral drug.This is especially true for the acute and/or topical therapies mostcommonly employed for the herpes simplex viruses.

The structure of this compound and its already proven drugabilityimplies that further improvements in the inhibitory potential againstthe herpesviruses should be attainable by standard medicinal chemistryapproaches.

Elimination in Mouse Liver Microsomes.

Table 3 below shows the elimination profile of compounds #191, #41, and#208. The table shows the half life, the intrinsic clearance, thehepatic clearance, extraction ratio, and methods of turnover. Severalcompounds tested in mouse liver microsomes had long half-lives, lowextraction ratios, and no evidence of non-P450 turnover (Table 3),suggesting their capacity to resist rapid degradation and elimination bythe liver.

TABLE 3 Polyoxygen heterocyclic compounds show favorable resistance toelimination in mouse liver microsomes. t ½ CL’int CL ‘hep Non-P450 turn-Compound (min) (mL/min/kg) (mL/min/kg) ER over 208 >120 −201 163 <0.1 No41 >120 17 14   0.16 No 191 >120 9 8   0.09 No

Summary.

The inventors here demonstrate that a polyoxygenated heterocyclecompound profoundly suppresses replication of HSV-1 and HSV-2 with nomeasurable toxicity in a short-term cell culture assay. Inhibition bythis primary screening hit is equal or superior to the approvedanti-herpesvirus drug acyclovir, particularly against HSV-2. Thus, theexisting compound may already be superior to the drugs that are used forHSV-2 infection, and improved efficacy can readily be envisioned throughstandard medicinal chemistry approaches. Its structural dissimilarity tothe nucleoside analogs implies a novel mode of action, suggesting itwould be a good candidate for combination therapy with the existinganti-herpesvirus drugs to improve efficacy of antiviral therapy.

3. HBV Activity

HBV activity of some of the compounds described herein is shown in Table4 below.

TABLE 4 HBV Activity Qualitative HBV Compound EC₅₀ CC₅₀ replicationNumber Compound (μM) (μM) inhibition assay 151

4.7 15 ** 153

4.1 10 ** 210

1.5 71 ** 208

0.69 15 ** 211

13 30 ** 49

ND 9 — **+ DNA suppression <25% and − DNA suppression >60% *+ DNAsuppression <50% of − DNA suppression ND   not determined

4. Synergistic Activity of HBV RNAseH with Nucleoside Analog Drug

Current nucleos(t)ide analog therapy for HBV has converted hepatitistype B from an implacably progressing illness to a controllable disease.However, patients are only very rarely cured, in part due to theincomplete inhibition of HBV replication. The inventors hypothesizedthat the novel RNaseH inhibitors would work synergistically with theexisting nucleos(t)ide analogs because the two classes of drugs targetphysically distinct active sites on the viral polymerase protein.Therefore, potential synergy between the RNAseH inhibitors and thenucleoside analog Lamivudine was analyzed using the Chou-Talalay method(Chou 2010). RNAseH inhibitors from two different chemical classes wereemployed, compound #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione], an HID,and #46 (3-thujaplicinol), an α-hydroxytropolone, were tested.Chou-Talaly analysis yields a combination index (CI). CI values <1.0indicate synergy, CIs of approximately 1.0 indicate additiveinteractions, and CI values >1.0 indicate antagonism. CI values arecalculated at various efficacy levels (EC₅₀, EC₇₅, EC₉₀, and EC₉₅), anda weighted CI value favoring higher efficacy levels is also generated.FIGS. 4A & 4B show the results of four experiments employing compound #1and three with compound #46. All experiments revealed synergisticinteractions between the RNAseH inhibitors and Lamivudine, and theweighted CI values were 0.70±0.1 for the HID compound #1 and 0.44±0.3for the α-hydroxytropolone #46. Therefore, RNAseH inhibitors actstrongly synergistically with an approved nucleos(t)ide analog drugagainst HBV. This demonstrates feasibility for employing RNAseHinhibitors in combination therapy with the nucleos(t)ide analogs duringHBV treatment.

5. RNAseH Inhibitor Sensitivity is Insensitive to High Genetic Variation

HBV has 8 genotypes differing in sequence by 8%. Genetic diversity inthe RNAseH domain is about 6%, which is easily high enough to modulateviral sensitivity to RNAseH inhibitors. Therefore, the inventors testedthe RNAseH inhibitors #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione], anHID, and #46 (β-thujaplicinol), an α-hydroxytropolone, for the abilityto inhibit variant RNAseHs. Twelve purified, patient-derived RNAseHenzymes (4 from each genotypes B, C and D) were tested with thecompounds at their respective IC₅₀ values in a biochemical RNAseH assay.FIGS. 5A & 5B demonstrate that the four genotype D enzymes eachinhibited the HBV RNAseH by about 50% at the compounds IC₅₀ values asexpected. Equivalent results were obtained for all 12 enzymes againstcompounds #1 and #46. Therefore, HBV's high genetic variation isunlikely to present a substantial barrier to drug development.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this disclosure havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the disclosure. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of thedisclosure as defined by the appended claims.

F. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   Alba et al., Genome Res., 11:43-54, 2001.-   Andrei and Snoeck, Curr Opin Infect Dis. 26:551-560, 2013.-   Aoki et al., Antimicrob Agents Chemother. 39:846-849, 1995.-   Ariyoshi et al., Cell 78:1063-1072, 1994.-   Arvin & Gilden, Varicella-Zoster Virus, 2015-2057, 2013.-   Balzarini et al., PLoS Pathog. 9:e1003456, 2013.-   Bernstein et al., Antiviral Res 92:386-388, 2011.-   Billamboz et al., J. Med. Chem. 54:1812-1824, 2011.-   Bogner et al., J Virol., 72:2259-2264, 1998.-   Bogner, Rev Med Virol., 12:115-127, 2002.-   Bokesch et al., J. Nat. Prod. 71, 1634-1636, 2008.-   Bortner et al., J Mol Biol. 231:241-250, 1993.-   Budihas et al., Nucleic Acids Res. 33, 1249-1256, 2005.-   Cai, et al., Antiviral Res., 108:48-55, 2014.-   Chen et al., J. Neurovirology 8:204-210, 2002.-   Choi et al., Antiviral Res. 55: 279-290, 2002.-   Chono et al., J. Antimicrob. Chemother. 65:1733-1741, 2010.-   Chou, et al., Cancer Res., 70:440-446, 2010.-   Chung et al., J. Med. Chem. 54, 4462-4473, 2011.-   Chung et al., Antimicrob. Agents Chemother. 54, 3913-3921, 2010.-   Clement et al., Int J Cancer 100:491-498, 2002.-   Coen, Antiviral Res, 15:287-300, 1991.-   Coen, Viral DNA polymerases, p. 495-523, 1996.-   Cohen, The New England journal of medicine 369:255-263, 2013.-   Damania & Cesarman, Kaposi's Sarcoma-Associated Herpesvirus,    2080-2128, 2013.-   De et al., Curr. Opin. Infect. Dis., 28:589-595, 2015.-   Decaro et al., The Veterinary clinics of North America. Small animal    practice 38:799-814, viii, 2008.-   Derse et al., J Biol Chem, 257:10251-10260, 1982.-   Di et al., Bioorg. Med. Chem. Lett. 20, 398-402, 2010.-   Didierjean et al., Antimicrob. Agents Chemother. 49, 4884-4894,    2005.-   Drew et al., Clin Diagn Virol. 1:179-185, 1993.-   Drew et al., J Infect Dis. 179:1352-1355, 1999.-   Drew et al., Am J Transplant 1:307-312, 2001.-   Dolan et al., J Virol 72:2010-2021, 1998.-   Duan et al., J Infect Dis, 200:1402-1414, 2009.-   Duan, et al. J Infect Dis, 198:659-663, 2008.-   Dyda et al., Science 266:1981-1986, 1994.-   Eberhard et al., Blood 114:3064-3073, 2009.-   Elion et al., Proc Natl Acad Sci USA, 74:5716-5720, 1977-   Fenner et al., Veterinary Virology, 2 ed. Academic Press, 1993.-   Field and Biron, Clin Microbiol Rev, 7:1-13, 1994.-   Field and Vere Hodge, Br Med Bull. 106:213-249, 2013.-   Fortier et al., Veterinary J. 186:148-156, 2010.-   Frank et al., Biol. Chem. 379:1407-1412, 1998.-   Frank et al., Proc. Natl. Acad. Sci. USA 95:12872-12877, 1998.-   Freed et al., “HIVs and their replication,” in: Knipe, D. M.,    Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A., Roizman,    B., Straus, S. E. (Eds.), FIELDS VIROLOGY. Lippincott Williams &    Wilkins, Philadelphia, pp. 2107-2185, 2007.-   Fuji et al., J. Med. Chem. 52, 1380-1387, 2009.-   Gao et al., Virology 249:460-470, 1998.-   Gaskell et al., Feline herpesvirus. Veterinary research 38:337-354,    2007.-   Gerelsaikhan et al., J. Virol. 70, 4269-4274, 1996.-   Gilbert et al., Canadian journal of public health=Revue canadienne    de sante publique, 2011.-   Gilbert, et al., Drug Resist Updat, 5:88-114, 2002.-   Gimeno, Vaccine 26 Suppl 3:C31-41, 2008.-   Goedken et al., J. Biol. Chem. 276, 7266-7271, 2001.-   Hanauske-Abel et al., PloS One 8:e74414. doi: 10.1371, 2013.-   Hanel et al., Mycoses 34 Suppl 1:91-93, 1991.-   Hanson et al., Viruses 3(11): 2160-2191, 2011.-   Higaki et al., Cornea 25(10 Suppl 1):S64-67, 2006.-   Himmel et al., ACS Chem. Biol. 1:702-712, 2006.-   Himmel et al., Structure 17: 1625-1635, 2009.-   Hirari, Current Topics in Microbiology and Immunology: Marek's    Disease, 2001.-   Hoffman et al., Cytometry 12:26-32, 1991.-   Horowitz et al., Journal of American college health: J of ACH    59:69-74, 2011.-   Hostomsky et al., Nulceases, vol. 2, 1993b.-   Hostomsky et al., “Ribonuclease H,” in: Linn, S. M., Lloyd, R. S.,    Roberts, R. J. (Eds.), Nulceases. Cold Spring Harbor Laboratory    Press, Plainview, N. Y., pp. 341-376, 1993a.-   Hostomsky et al., Structure 3:131-134, 1993c.-   Hu et al., Antiviral Res 99:221-229, 2013.-   Hwang and Bogner, J Biol Chem., 277:6943-6948, 2002.-   Imai et al., J Infect Dis. 189:611-615, 2004.-   James et al., Antiviral Res 83:207-213, 2009.-   Johnston et al., Lancet 379:641-647, 2012.-   Kamali et al., Sexually transmitted infections 75:98-102, 1999.-   Katayanagi et al., Nature 347: 306-309, 1990.-   Keck et al., J. Biol. Chem. 273, 34128-34133, 1998.-   Kim et al., In Vivo 25:887-893, 2011.-   Kimberlin, Seminars in perinatology 31:19-25, 2007.-   Kirschberg et al., J. Med. Chem. 52:5781-5784, 2009.-   Klarmann et al., AIDS Rev 4: 183-194, 2002.-   Klumpp et al., Nucleic Acids Res. 31, 6852-6859, 2004.-   Klumpp and Mirzadegan, Curr. Pharm. Des 12:1909-1922, 2006.-   Knipe and Spang J Virol, 43:314-324, 1982.-   Ko et al., PLoS One 6:e27844. doi: 10.1371, 2011.-   Komatsu et al., Antiviral Res 101:12-25, 2014.-   Korom et al., J Virol, 87:5882-5894, 2013.-   Kwun et al., J. Gen. Virol. 82, 2235-2241, 2001.-   Lai et al., Structure 8:897-904, 2000.-   Levin et al., Clin Infect Dis, 39 Suppl 5:S248-257, 2004.-   Li et al., Mol. Biol. Evol. 12:657-670, 1999.-   Lima et al., Methods Enzymol. 341:430-440, 2001.-   Linden et al., Faseb J., 17:761-763, 2003.-   Liu et al., J. Biol. Chem. 281:18193-18200, 2006.-   Longnecker et al., Epstein-Barr Virus, 1898-1959, 2013.-   Looker, et al., Bull. World Health Organ., 86:805-812, A, 2008.-   Lu, et al., Antimicrob. Agents Chemother., 59(2):1070-1079, 2015.-   Luzuriaga & Sullivan, N Eng J Med. 362:1993-2000, 2010.-   Manicklal et al., Clinical Microbiology Rev. 26:86-102, 2013.-   Marcellin et al., N. Engl. J. Med. 359: 2442-2455, 2008.-   Marchand, Tchesnokov, and Gotte. J Biol Chem 282: 3337-3346, 2007.-   Marfori et al., J Clin Virol. 38:120-5, 2007.-   Masaoka, et al., Biochemistry, 55(5):809-819, 2016.-   McDermott et al., J Virol, 51:747-753, 1984.-   Mettenleiter et al., Virus Res. 143:222-234, 2009.-   Mohni et al., J. Virol. 85:12241-12253, 2011.-   Morfin and Thouvenot, J Clin Virol., 26:29-37, 2003.-   Morrison and Knipe, Virology, 220:402-413, 1996.-   Nakagawa and Tayama, Chem Biol Interact, 116:45-60, 1998.-   Nandi et al., Animal health research reviews/Conference of Research    Workers in Animal, 2009.-   Nauwynck et al., Veterinary Res. 38:229-241, 2007.-   Nimonkar and Boehmer, J Biol Chem. 278:9678-9682, 2003.-   Nowotny et al., Cell 121: 1005-1016, 2005.-   Nowotny, EMBO Rep. 10:144-151, 2009.-   Obasi et al., J. Infectious Dis. 179:16-24, 1999.-   Parker et al., EMBO J. 23: 4727-4737, 2004.-   Pellet & Roizman, Herpesviridae 1802-1822, 2013.-   Pelosi et al., Adv Exp Med Biol, 312:151-158, 1992.-   Pena et al., J. Clin. Microbiol. 48:150-153, 2010.-   Popovic et al., J. Thrombosis Thrombolysis 33:160-172, 2012.-   Potenza et al., Protein Expr. Purif 55: 93-99, 2007.-   Quenelle et al., Antivir Chem Chemother 22:131-137, 2011.-   Quinlan et al., Cell 36:857-868, 1984.-   Reyes et al., Arch Intern Med, 163:76-80, 2003.-   Roizman et al., Herpes Simplex Viruses, 1823-1897, 2013.-   Rosen et al., Int J Dermatol., 36:788-792, 1997.-   Scheffczik et al., Nucleic Acids Res., 30:1695-1703, 2002.-   Schmit and Boivin, J Infect Dis, 180:487-490, 1999.-   Scholz et al., Nucleic Acids Res., 31:1426-1433, 2003.-   Schumacher et al., PLoS Pathog. 8:e1002862, 2012.-   Sehgal, Br J Dermatol. 95:83-88, 1976.-   Selvarajan et al., J. Virol. 87:7140-7148, 2013.-   Semenova et al., Mol. Pharmacol. 69:1454-1460, 2006.-   Shaw-Reid et al., J. Biol. Chem. 278, 2777-2780, 2003.-   Smith & Roberts, J. ACH 57:389-394, 2009.-   Smith et al., J. Virol. 68, 5721-5729, 1994.-   Smith, Veterinary J. 153:253-268, 1997.-   Snydman, Clin Infect Dis. 47:883-884, 2008.-   Song et al., Science 305: 1434-1437, 2004.-   Su et al., J. Virol. 84:7625-7633, 2010.-   Takada et al., J. Nat. Prod. 70, 1647-1649, 2007.-   Tavis and Lomonosova, Antiviral Res., 118:132-138, 2015.-   Tavis et al., PLoS Pathogens 9:e1003125, 2013.-   Tavis, et al., Antimicrobial Agents and Chemotherapy,    58(12):7451-7461, 2014.-   Tyring et al., J. Infect. Dis. 205:1100-1110, 2012.-   Wald et al., N Engl J Med. 370:201-210, 2014.-   Wang et al., J. Virol. 79:14079-14087, 2005.-   Wang et al., J Clin Virol, 52:107-112, 2011.-   Weizman & Weller, Interactions between HSV-1 and the DNA damage    response, p. 257-268, 2011.-   Weller & Coen, Cold Spring Harbor Perspectives in Biology 4:a013011,    2012.-   Weller and Kuchta, Expert Opin Ther Targets, 17:1119-1132, 2013.-   Wendeler et al., ACS Chem. Biol. 3, 635-644, 2008.-   Williams et al., Bioorg. Med. Chem. Lett. 20:6754-6757, 2010.-   Xu et al., JAMA 296:964-973, 2006.-   Yamanishi et al., Human Herpesviruses 6 and 7, p. 2058-2079, 2013.-   Yan et al., MBio 5:e01318-14, 2014.-   Yang et al., Science 249: 1398-1405, 1990.-   Yang and Steitz, Structure, 3, 131-134, 1995.-   Yao et al., Antimicrob Agents Chemother. 58:2807-2815, 2014.-   Zhou et al., Int J Cancer 127:2467-2477, 2010.-   Zhou et al., J Virol. 88:11121-11129, 2014.-   Zhu et al., J. Virol. 84:7459-7472, 2010.

1. A method of inhibiting a herpesvirus or hepatitis B virus nucleicacid metabolism enzyme comprising contacting said enzyme with a compoundhaving the formula:

or a compound of the formula:

wherein: R₁ is aryl_((C≤12)), aralkyl_((C≤18)), heteroaryl_((C≤12)),alkylamino_((C≤12)), dialkylamino_((C≤12)), arylamino_((C≤12)),diarylamino_((C≤12)), aralkylamino_((C≤18)), diaralkylamino_((C≤18)), ora substituted version of any of these groups; R₂ is hydrogen,alkyl_((C≤8)), or substituted alkyl_((C≤8)); and R₃ is hydrogen, amino,carboxyl, cyano, halo, hydroxy, nitro, hydroxysulfonyl, orsulfonylamine; or alkyl_((C≤8)), aryl_((C≤8)), acyl_((C≤8)),alkoxy_((C≤8)), acyloxy_((C≤8)), amido_((C≤8)), or substituted versionof any of these groups; X₂ is hydrogen or —C(O)R_(a), wherein: R_(a) ishydroxy, alkoxy_((C≤8)), or substituted alkoxy_((C≤8)); or a compound ofthe formula:

wherein: R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; R₅and R₈ are each independently hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and R₇ is aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; ora compound of the formula:

wherein: R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups;R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and R₁₁ ishydrogen or Y₁—O—X₁—OR₁₂; wherein: Y₁ is alkanediyl_((C≤8)) orsubstituted alkanediyl_((C≤8)); X₁ is arenediyl_((C≤12)),heteroarenediyl_((C≤12)), or a substituted version of either of thesegroups; R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), ora substituted version of any of these groups; or a pharmaceuticallyacceptable salt or tautomer thereof.
 2. The method of claim 1, whereinthe compound is further defined as:

wherein: R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; R₅and R₈ are each independently hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and R₇ is aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; oror a pharmaceutically acceptable salt or tautomer thereof.
 3. The methodof claim 1, wherein the compound is further defined as:

wherein: R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups;R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and R₁₁ ishydrogen or Y₁—O—X₁—OR₁₂; wherein: Y₁ is alkanediyl_((C≤8)) orsubstituted alkanediyl_((C≤8)); X₁ is arenediyl_((C≤12)),heteroarenediyl_((C≤12)), or a substituted version of either of thesegroups; R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), ora substituted version of any of these groups; or a pharmaceuticallyacceptable salt or tautomer thereof.
 4. The method of claim 1, whereinthe compound is further defined as:

or a pharmaceutically acceptable salt or tautomer thereof.
 5. The methodof claim 1, wherein the compound is further defined as:

or a pharmaceutically acceptable salt or tautomer thereof.
 6. (canceled)7. The method of claim 1, further comprising contacting said enzyme witha second inhibitor of said enzyme.
 8. (canceled)
 9. The method of claim7, wherein said enzyme is located in a cell.
 10. (canceled)
 11. Themethod of claim 9, wherein said cell is located in a living subject. 12.The method of claim 11, wherein said subject is a vertebrate infectedwith a herpesvirus.
 13. The method of claim 12, wherein said compound isadministered intravenously, intra-arterially, orally, buccally, nasally,ocularly, rectally, vaginally, topically, intramuscularly,intradermally, cutaneously or subcutaneously.
 14. The method of claim12, wherein said subject is further administered a secondanti-herpesvirus therapy.
 15. The method of claim 14, wherein saidsecond anti-herpesvirus therapy is foscarnet or a nucleoside analog. 16.The method of claim 15, wherein said nucleoside analog is acyclovir,famciclovir, valaciclovir, penciclovir, or ganciclovir. 17.-18.(canceled)
 19. The method of claim 1, wherein said subject haspreviously received a first-line anti-herpesvirus therapy.
 20. Themethod of claim 19, wherein said herpesvirus has developed resistance tosaid first-line anti-herpesvirus therapy.
 21. The method of claim 1,wherein said herpevirus is selected from a human alpha herpesvirus, ahuman beta herpesvirus or a human gamma herpesvirus. 22.-51. (canceled)52. A method of inhibiting replication of a hepatitis B virus comprisingcontacting the virus with an effective amount of a compound orcomposition further defined as:

wherein: R₁ is aryl_((C≤12)), aralkyl_((C≤18)), heteroaryl_((C≤12)),alkylamino_((C≤12)), dialkylamino_((C≤12)), arylamino_((C≤12)),diarylamino_((C≤12)), aralkylamino_((C≤18)), diaralkylamino_((C≤18)), ora substituted version of any of these groups; R₂ is hydrogen,alkyl_((C≤8)), or substituted alkyl_((C≤8)); and R₃ is hydrogen, amino,carboxyl, cyano, halo, hydroxy, nitro, hydroxysulfonyl, orsulfonylamine; or alkyl_((C≤8)), aryl_((C≤8)), acyl_((C≤8)),alkoxy_((C≤8)), acyloxy_((C≤8)), amido_((C≤8)), or substituted versionof any of these groups; X₂ is hydrogen or —C(O)R_(a), wherein: R_(a) ishydroxy, alkoxy_((C≤8)), or substituted alkoxy_((C≤8)); or a compound ofthe formula:

wherein: R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; R₅and R₈ are each independently hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and R₇ is aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; ora compound of the formula:

wherein: R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups;R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and R₁₁ ishydrogen or Y₁—O—X₁—OR₁₂; wherein: Y₁ is alkanediyl_((C≤8)) orsubstituted alkanediyl_((C≤8)); X₁ is arenediyl_((C≤12)),heteroarenediyl_((C≤12)), or a substituted version of either of thesegroups; R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), ora substituted version of any of these groups; or a pharmaceuticallyacceptable salt thereof. 53.-55. (canceled)
 56. The method of claim 52,wherein the method is sufficient to treat an infection of a hepatitis Bvirus.
 57. A method of treating an infection of a hepatitis B virus in apatient comprising administering a therapeutically effective amount of acompound or composition further defined as:

wherein: R₁ is aryl_((C≤12)), aralkyl_((C≤18)), heteroaryl_((C≤12)),alkylamino_((C≤12)), dialkylamino_((C≤12)), arylamino_((C≤12)),diarylamino_((C≤12)), aralkylamino_((C≤18)), diaralkylamino_((C≤18)), ora substituted version of any of these groups; R₂ is hydrogen,alkyl_((C≤8)), or substituted alkyl_((C≤8)); and R₃ is hydrogen, amino,carboxyl, cyano, halo, hydroxy, nitro, hydroxysulfonyl, orsulfonylamine; or alkyl_((C≤8)), aryl_((C≤8)), acyl_((C≤8)),alkoxy_((C≤8)), acyloxy_((C≤8)), amido_((C≤8)), or substituted versionof any of these groups; X₂ is hydrogen or —C(O)R_(a), wherein: R_(a) ishydroxy, alkoxy_((C≤8)), or substituted alkoxy_((C≤8)); or a compound ofthe formula:

wherein: R₄ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; R₅and R₈ are each independently hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); R₆ is hydrogen, hydroxy, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and R₇ is aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups; ora compound of the formula:

wherein: R₉ is alkyl_((C≤12)), aryl_((C≤12)), aralkyl_((C≤12)),heteroaryl_((C≤12)), or a substituted version of any of these groups;R₁₀ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and R₁₁ ishydrogen or Y₁—O—X₁—OR₁₂; wherein: Y₁ is alkanediyl_((C≤8)) orsubstituted alkanediyl_((C≤8)); X₁ is arenediyl_((C≤12)),heteroarenediyl_((C≤12)), or a substituted version of either of thesegroups; R₁₂ is aryl_((C≤12)), aralkyl_((C≤12)), heteroaryl_((C≤12)), ora substituted version of any of these groups; or a pharmaceuticallyacceptable salt thereof. 58.-59. (canceled)
 60. The method of claim 1,wherein the method is sufficient to inhibit viral replication.